This work was supported by the National Natural Science Foundation of China (21435008 and 21327006 to H.W.), and the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB14030200 to H.W.).

1. Preparations
<ol>
	<li>Prepare a solution of 1.0 mM PD0325901 (MEK inhibitor) and 3.0 mM CHIR99021 (GSK3 inhibitor).
	<ol>
		<li>Weigh 2 mg of PD0325901 and add 4.15 mL of DMSO in an amber glass vial.</li>
		<li>Weigh 2 mg of CHIR99021 and add 1.43 mL of DMSO in an amber glass vial.</li>
		<li>Following reconstitution, store aliquots (50 &#181;L/tube in 200 &#181;L PCR tubes) at -20 &#176;C and protected from light. Remove a tube containing the frozen stock from a freezer and thaw at room temperature before use.</li>
	</ol>
	</li>
	<li>Weigh 0.0176 g of Vc into 1 mL of a centrifuge tube and reconstitute it with 1 mL of sterile water to a final concentration of 100 mM prior to use.</li>
	<li>Inactivate FBS: incubate 500 mL of FBS in a water bath at 56 &#176;C for 30 min.</li>
	<li>Prepare 600 mL of basic medium for culturing mouse ES cells.
	<ol>
		<li>Use a bottle of DMEM/high glucose medium (500 mL) and remove 40 mL.</li>
		<li>Supplement 460 mL of DMEM/high glucose medium with 20% inactivated FBS (120 mL), 1,000 U/mL LIF (60 &#181;L of 107 U/mL stock solution), 0.1 mM non-essential amino acids (NEAA) (6 mL of 10 mM stock solution), 1 mM sodium pyruvate (6 mL of 100 mM stock solution), 2 mM L-glutamine (6 mL of 200 mM stock solution), 0.1 mM &#946;-mercaptoethanol (600 &#181;L of 100 mM stock solution), and 33 IU/mL penicillin and 33 &#181;g/mL streptomycin (2 mL of penicillin-streptomycin mixed solution (10,000 IU/mL penicillin and 10,000 &#181;g/mL streptomycin)). Define the basic medium as serum medium.</li>
		<li>Pipette 50 &#181;L of stock solution of PD0325901 (1.0 mM) and Vc (100 mM), respectively, into 50 mL of the serum medium (final concentration: 1.0 &#181;M PD0325901 and 100 &#181;M Vc), and define the medium as Vc/PD0325901 medium. 
		NOTE: Prepare the Vc/PD0325901 medium fresh before use due to the instability of Vc.</li>
		<li>Using a pipette, transfer 50 &#181;L of stock solution of PD0325901 (1.0 mM) and CHIR99021 (3.0 mM), respectively, into 50 mL of the serum medium (final concentration: PD0325901 1.0 &#181;M and CHIR99021 3.0 &#181;M), and define the medium as 2i medium.</li>
	</ol>
	</li>
	<li>Prepare the gelatin-coated dishes.
	<ol>
		<li>Prepare the 0.1% gelatin solution: dissolve 0.3 g of gelatin into 300 mL of ultrapure water in a glass reagent bottle with a cap and sterilize in an autoclave at 121 &#176;C for 20 min. Store the sterile solution at room temperature.</li>
		<li>Incubate the cell culture dishes (6 cm diameter) with 2 mL of 0.1% gelatin solution for 15 min at room temperature and then aspirate the gelatin. Dry the dishes in air and use them within 12 h.</li>
	</ol>
	</li>
	<li>Prepare 10 mL of mouse ES cell freezing medium in a 15 mL centrifuge tube: 90% FBS (9 mL) and 10% DMSO (1 mL). Use the medium within 1 day.</li>
	<li>Prepare a freezing container: add 100% isopropyl alcohol to the fill line of the freezing container, and discard the old isopropyl alcohol. Add new isopropyl alcohol directly after every fifth use.</li>
	<li>Make the enzymatic digestion buffer: weigh 1.2114 g of Tris powder into 100 mL of ultrapure water to prepare 100 mM Tris solution and add concentrated HCl solution (about 12 M) into the Tris solution to adjust the pH to 7.6 (about 0.6 mL of concentrated HCl solution).</li>
	<li>Prepare 50 mL of radio immunoprecipitation assay buffer (RIPA buffer): 20 mM Tris, pH 7.4, 1 mM MgCl2, 150 mM NaCl, 20% glycerol, 0.5% NP40, 1 mM EDTA, 1 mM EGTA. Supplement with 1 mM DTT, 1X protease inhibitor cocktail, and 1 mM PMSF prior to use. Once the PMSF is added, use the buffer within 30 min.</li>
</ol>
2. Grow Mouse ES Cells on Gelatin-coated Dishes
<ol>
	<li>Pre-warm mouse ES cells (wild type, 129 SvEv) culture medium, trypsin, and phosphate buffered solution (PBS) in a water bath at 37 &#176;C.</li>
	<li>Passage the cells. Aspirate the medium completely from the dish and pipette 2 mL of PBS to the dishes to rinse the cells.
	<ol>
		<li>Remove the PBS with a pipette and wash the cells again with 2 mL of PBS.</li>
		<li>Remove the PBS and add 0.3 mL of trypsin-EDTA (0.25%) to each dish.</li>
		<li>Cover the whole dish with the trypsin quickly and then remove it immediately.</li>
		<li>Put the dish in an incubator at 37 &#176;C for 1 min to detach the cells from the dish. Take out the dishes from the incubator and add 2 mL of pre-warmed serum medium to terminate the action of trypsin.</li>
		<li>Dissociate the cell colonies into a single cell suspension by resuspending with a pipette (5 mL pipette tip) 10 times.</li>
	</ol>
	</li>
	<li>Transfer 150 &#181;L of 2 mL of the cell solution (about 190,000 cells by counting with a hemocytometer) into a new gelatin-coated dish, and supplement with 3 mL of freshly made Vc/PD0325901 medium per 6 cm culture dish. Culture the cells in a 37 &#176;C incubator with 5% CO2.</li>
	<li>Every 24 h while incubating, remove the old culture medium and add 3 mL of fresh Vc/PD0325901-medium into the culture dish due to the instability of Vc. Expect 70&#8211;80% confluency after 2&#8211;3 days.</li>
	<li>After reaching 70-80% confluency, passage the cells and continue to culture them as described in steps 2.2&#8211;2.4.</li>
</ol>
3. Freeze and Thaw Mouse ES Cells
<ol>
	<li>Freeze mouse ES cells.
	<ol>
		<li>Detach the cells from the dishes with trypsin by following step 2.2.</li>
		<li>Transfer the cells into a 15 mL plastic tube and centrifuge at 800 x g and room temperature for 3 min.</li>
		<li>Dump the supernatant gently into a beaker and resuspend the cell pellet with 1 mL of freshly made freezing medium. Prepare the freezing medium 30 min prior to this step to ensure that it is room temperature before use.</li>
		<li>Transfer the cells in freezing medium to a 1.8 mL microcentrifuge tube with a 1 mL pipette and place the microcentrifuge tube into a freezing container. Add isopropyl alcohol to the fill line of the freezing container and keep it at room temperature before using.</li>
		<li>Leave the freezing containers overnight in a -80 &#176;C freezer.</li>
		<li>Transfer the microcentrifuge tubes to a liquid nitrogen container for up to 2&#8211;3 years. 
		NOTE: Do not keep cells in the freezing medium for a long time and quickly transfer the cells to a -80 &#176;C freezer due to the toxicity of DMSO.</li>
	</ol>
	</li>
	<li>Thaw mouse ES cells.
	<ol>
		<li>Pre-warm 50 mL of serum medium in a 37 &#176;C water bath.</li>
		<li>Take out a cell-containing vial from the liquid nitrogen container and immerse the capped vial in a 37 &#176;C water bath. Shake it quickly in the water bath to thaw. Transfer the cells into a 15 mL tube with a 1 mL pipette. Add 2 mL of pre-warmed serum culture medium into the tube to dilute the freezing medium and then centrifuge at 800 x g and room temperature for 3 min.</li>
		<li>Remove the supernatant and gently resuspend the cell pellets with 3 mL of fresh Vc/PD0325901 medium using a 5 mL pipette.</li>
		<li>Transfer the cells from the 15 mL tube to a gelatin-coated dish and culture the cells for 24 h. Culture the cells again as described in Step 2.</li>
	</ol>
	</li>
</ol>
4. Extract Total Protein from Cells
<ol>
	<li>Rinse the collected cells with 1 mL of PBS and then centrifuge at 800 x g and room temperature for 3 min.</li>
	<li>Aspirate the supernatant and resuspend the cell pellet thoroughly with RIPA buffer supplemented with DTT, protease inhibitor cocktail, and PMSF by pipetting gently. The volume of RIPA buffer is about 5x that of the cell pellet. 
	NOTE: Carry out the cell resuspension on ice.</li>
	<li>Keep the cells in RIPA buffer for 30 min on ice and centrifuge at 13,000 x g and 4 &#176;C for 5 min.</li>
	<li>Transfer the supernatant to a new tube and store aliquots at -80 &#176;C.</li>
	<li>Determine the concentration of the protein with a Bradford protein assay kit.</li>
</ol>
5. Extract DNA from Cells and Digest DNA into a Single Nucleoside Using Enzymes
<ol>
	<li>Collect the cells with trypsin as described in steps 3.1.1 and 3.1.2.</li>
	<li>Perform the DNA extraction with a genomic DNA purification kit following the manufacturer&#39;s instructions.</li>
	<li>Store the extracted DNA in 1-mL centrifuge tubes. Add 100 &#181;L of ultrapure water into the centrifuge tube, and dissolve the DNA by pipetting approximately 15 times. Determine the concentration and evaluate the quality of DNA by the measurement of the absorbance at 260 nm and 280 nm15. The DNA concentration is about 500 ng/&#181;L.</li>
	<li>Digest 5 &#181;g of DNA in a 50 &#181;L solution system: add 5 &#181;L of 100 mM Tris-HCl solution, pH 7.6 (final concentration: 10 mM), 2 U calf intestinal phosphatase, 1 U DNase I, 0.005 U snake venom phosphodiesterase I, and 5 &#181;g of DNA (calculate the added volume according to the measured DNA concentration, ~10 &#181;L) into a centrifuge tube and increase the volume of solution to 50 &#181;L with ultrapure water. Incubate the mixture at 37 &#176;C overnight. Centrifuge the digested DNA solution at 1,000 g and room temperature for 1 min to collect the solution to the bottom of tubes.</li>
	<li>Transfer the digested DNA solution from the centrifuge tubes into ultra-filtration tubes (a molecule weight cutoff: 3 kDa) and centrifuge at 13,000 g and 4 &#176;C for 30 min to remove the digestion enzymes.</li>
	<li>Prepare the samples for 5mC and 5hmC analysis.
	<ol>
		<li>For 5hmC analysis: pipet 36 &#181;L of filter solution to a new centrifuge tube (1 mL) and add 4 &#181;L of 5&#39;-(hydroxymethyl-d3)-2&#39;-deoxycytidine ([D3]-5hmC) with the final concentration of 3 nM.</li>
		<li>For 5mC analysis: add 196 &#181;L of ultrapure water into a new centrifuge tube and pipet 4 &#181;L of filter solution to the tube (50-fold dilution), due to the high density of 5mC in genomic DNA. Transfer 36 &#181;L of the diluted solution to a new centrifuge tube and add 4 &#181;L of (5&#39;-(methyl-d3)-2&#39;-deoxycytidine ([D3]-5mC) with the final concentration of 50 nM. Use [D3]-5hmC and [D3]-5mC as the internal standard to calibrate 5hmC and 5mC, respectively.</li>
	</ol>
	</li>
	<li>Transfer the sample solution to measuring tubes and store at 4 &#176;C. Analyze 5mC and 5hmC with ultra-high-performance liquid chromatography-triple quadrupole mass spectrometry with multiple-reaction monitoring (UHPLC-MS/MS (MRM)) as described in a previous report15.</li>
</ol>
6. Analyze 5mC and 5hmC Using UHPLC-MS/MS15
<ol>
	<li>Set up various parameters for analyzing 5mC and 5hmC using the UHPLC-MS software.
	<ol>
		<li>Establish a new method. Open the software and click &#34;File | New | Method&#34;. Exhibit the message box of &#34;Method Editor&#34; with the content &#34;Do you want to save the changes for the current method&#34; and click &#34;No&#34;.</li>
		<li>Build the parameters of the sampler. Click &#34;Sampler | Setup | Injection&#34;. Select &#34;Standard injection&#34; and input &#34;5 &#181;L at Injection volume&#34;.</li>
		<li>Build the parameters of the pump in UHPLC.
		<ol>
			<li>Click &#34;BinPump2 | Setup&#34; to build the parameters of the pump. Set up &#34;Flow&#34; at &#34;0.25 mL/min&#34; and &#34;Stop time&#34; at &#34;15 min&#34;.</li>
			<li>Set up &#34;Solvent B at 5%&#34; and &#34;Pressure limits&#34; at &#34;Max 600 bar&#34;.</li>
			<li>Click &#34;BinPump2 | Timetable&#34; to build the isocratic elution parameters for 5mC analysis. Click &#34;Append&#34; to add a row. Input &#34;0.00&#34; at &#34;Time&#34; and &#34;5.0&#34; at &#34;B%&#34;. Click &#34;Append&#34; once more to add a new row. Input &#34;15.00&#34; at &#34;Time&#34; and &#34;5.0&#34; at &#34;B%&#34;.</li>
			<li>Click &#34;BinPump2 | Timetable&#34; to build the gradient elution parameters for 5hmC analysis. Click &#34;Append to add a row. Input 0.00 at Time and 5.0 at &#34;B%&#34;. Click &#34;Append&#34; once more to add a new row. Input &#34;3.00&#34; at &#34;Time&#34; and &#34;5.0&#34; at &#34;B%&#34;. Click &#34;Append&#34; for another 6 times to add 6 rows. Input &#34;3.01&#34; at &#34;Time&#34; and &#34;15.0&#34; at &#34;B%&#34;, &#34;6.00&#34; at &#34;Time&#34; and &#34;15.0&#34; at &#34;B%&#34;, &#34;6.01&#34; at &#34;Time&#34; and &#34;100&#34; at &#34;B%&#34;, &#34;10.00&#34; at &#34;Time&#34; and &#34;100.0&#34; at &#34;B%&#34;, &#34;10.01&#34; at &#34;Time&#34; and &#34;5.0&#34; at &#34;B%&#34;, &#34;15.00&#34; at &#34;Time&#34; and &#34;5.0&#34; at &#34;B%&#34; in sequence. 
			NOTE: Perform the analysis of 5mC and 5hmC individually due to the different separation conditions of UHPLC.</li>
		</ol>
		</li>
		<li>Build the parameters of the temperature of the column compartment (TCC) in UHPLC. 
		Click &#34;TCC | Setup | temperature (Left)&#34; and input &#34;30 &#176;C&#34;.</li>
		<li>Establish the parameters of the QQQ-MS/MS.
		<ol>
			<li>Click &#34;MS QQQ | Stop time | No limit/As pump&#34;, &#34;Ion Source | ESI&#34;, &#34;Time filtering | Peak width: 0.07 min&#34;. Set up &#34;Time segments: Start time: 0&#34;; &#34;Scan Type: MRM&#34;; &#34;Div Value: To MS&#34;; &#34;Delta EMV (+): 200&#34; and &#34;Delta EMV (-): 0&#34;.</li>
			<li>Build the parameters of monitoring the transitions of 5mC, D3-5mC, dC, 5hmC, and D3-5hmC
			<ol>
				<li>Click &#34;MS QQQ | Acquisition | Scan segments: Dwell: 90&#34;; &#34;Fragmentor: 90&#34;; &#34;Collision Energy: 5&#34;; &#34;Cell Accelerator Voltage: 4&#34; and &#34;Polarity: Positive&#34;.</li>
				<li>Input &#34;5mC&#34; at &#34;Compound Name&#34;, &#34;242&#34; at &#34;Precursor Ion&#34;, &#34;126&#34; at &#34;Product Ion&#34; for 5mC analysis. Click the right button and select &#34;Add row&#34; to add a new row. Input &#34;D3-5mC&#34; at &#34;Compound Name&#34;, &#34;245&#34; at &#34;Precursor Ion&#34;, &#34;129&#34; at &#34;Product Ion&#34; for D3-5mC analysis.</li>
				<li>Supplement another three rows using the same mode. Input &#34;dC&#34; at &#34;Compound Name&#34;, &#34;228&#34; at &#34;Precursor Ion&#34;, &#34;112&#34; at &#34;Product Ion&#34; for dC analysis. Input &#34;5hmC&#34; at &#34;Compound Name&#34;, &#34;258&#34; at &#34;Precursor Ion&#34;, &#34;142&#34; at &#34;Product Ion&#34; for 5hmC analysis. Input D3-5hmC at Compound Name, &#34;261&#34; at &#34;Precursor Ion&#34;, &#34;145&#34; at &#34;Product Ion&#34; for D3-5hmC analysis.</li>
			</ol>
			</li>
			<li>Build the parameters of the gas source. Click &#34;MS QQQ | Source | Source parameters: Gas Temp: 300 &#176;C&#34;; &#34;Gas Flow: 11 L/min&#34;; &#34;Nebulizer: 15 psi&#34;; &#34;Positive-Capillary: 3500 V&#34;.</li>
			<li>Save the proposed method. Click &#34;MS QQQ | Instrument | Save method As&#34;. Select a pathway for the proposed method by &#34;Directory&#34; and give a name for the method by inputting the content in &#34;Method&#34;, for example: 5mC and 5hmC analysis.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>UHPLC separation and MS/MS analysis of mononucleosides
	<ol>
		<li>Prepare the mobile phase for 5mC and cytosine (C) analysis: form the mobile phase of solution A and B. Solution A: 500 mL of ultrapure water with 500 &#181;L of formic acid (final concentration: 0.1%), and solution B: 500 mL of 100% methanol. Mix solution A and B at 95:5 (v:v) as the mobile phase to elute 5mC and C (set in step 6.1.3.3). Set the flow rate at 0.25 mL/min (set in step 6.1.3.1).</li>
		<li>Prepare the mobile phase by solvent A and B for 5hmC analysis. Solvent A is 2.0 mM NH4HCO3 aqueous solution (pH 9.0) (500 mL), and solvent B is 100% methanol (500 mL). Separate 5hmC by an optimized gradient elution: 0-3 min, 5.0% B and 95% A (v:v); 3-6 min, 15.0% B and 85% A; 6-10 min, 100% B; 10-15 min, 5.0% B and 95% A (set in step 6.1.3.4). Set the flow rate at 0.25 mL/min (set in step 6.1.3.1).</li>
	</ol>
	</li>
	<li>Utilize electrospray MS/MS with MRM mode (set in step 6.1.5.1) to analyze the elution from the column and adopt the positive ion mode (set in step 6.1.5.2.1).
	<ol>
		<li>Monitor the transitions: 5mC, m/z 242&#8594;126 (collision energy, 5 eV); [D3]-5mC, m/z 245&#8594;129 (5 eV); dC, m/z 228&#8594;112 (5 eV); 5hmC, m/z 258&#8594;142 (5 eV); [D3]-5hmC, m/z 261&#8594;145 (5 eV) (set in step 6.1.5.2.2).</li>
		<li>Set the capillary and fragment voltages at +3,500 V (set in step 6.1.5.3) and 90 V (set in step 6.1.5.2.1), respectively.</li>
		<li>Set the injection volume at 5 &#181;L and analyze each sample 3 times (set in step 6.1.2). Employ the corresponding standard curves to evaluate the amount of 5mC and 5hmC.
		<ol>
			<li>Establish the standard curve of 5mC: transfer 1&#8211;50 nM (1, 5, 10, 20, 50 nM, final concentration) standard solution of 5mC into centrifuge tubes and add 50 nM [D3]-5mC to tubes. Supplement the total volume to 50 &#181;L. Perform the analysis of standard 5mC following the instructions for the 5mC sample (step 6).</li>
			<li>Build the standard curve of 5hmC: transfer 1&#8211;20 nM (1, 2, 5, 10, 20 nM, final concentration) standard solution of 5hmC to centrifuge tubes and add 3 nM [D3]-5hmC to the tubes. Supplement the total volume to 50 &#181;L. Perform the analysis of standard 5hmC following the instructions for the 5hmC sample (step 6).</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
7. Analyze the Statistical Significance
<ol>
	<li>Perform the statistical analysis using software.
	<ol>
		<li>Open the software and select &#34;Column&#34; in &#34;New table &#38; graph&#34;. Set the parameters as follows: &#34;Sample data | Start with an empty data table&#34;; &#34;Choose a graph-the first mode&#34;; &#34;Graphing replicates or error bars | Plot | Mean with SEM&#34;.</li>
		<li>Click &#34;Create&#34; to input the three replicates of Control and Vc/PD0325901-treated group in the &#34;A&#34; and &#34;B&#34; line, respectively. Select the six data and click &#34;Analyze&#34; in the &#34;Analysis&#34;.</li>
		<li>Select &#34;t tests (and nonparametric tests)&#34; in &#34;Column Analyses&#34; and click &#34;OK&#34; to enter the next interface.</li>
		<li>Set the parameters as follows: &#34;Choose test | Test name | Unpaired t test; Options | Two-tailed&#34;; &#34;Confidence Intervals: 95%&#34;; &#34;Significant digits | Show 4 significant digits&#34;. Click &#34;OK&#34; to acquire the p value between the Control and Vc/PD0325901 treatment.</li>
	</ol>
	</li>
	<li>Evaluate the statistical significance between the control and the experimental groups employing the two-tailed and unpaired Student&#39;s t-test16. 
	NOTE: p &#60;0.05 denotes the difference possessing statistical significance. *p &#60;0.05, **p &#60;0.01, ***p &#60;0.001.</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minocycline+modulates+NF&#954;B+phosphorylation+and+enhances+antimicrobial+activity+against+Staphylococcus+aureus+in+mesenchymal+stromal/stem+cells.">Minocycline modulates NF&#954;B phosphorylation and enhances antimicrobial activity against Staphylococcus aureus in mesenchymal stromal/stem cells.</a> Stem Cell Res. Ther. 8 (1), 171 (2017).</li><li>Blaschke, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitamin+C+induces+Tet-dependent+DNA+demethylation+and+a+blastocyst-like+state+in+ES+cells.">Vitamin C induces Tet-dependent DNA demethylation and a blastocyst-like state in ES cells.</a> Nature. 500 (7461), 222-226 (2013).</li><li>Tahiliani, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conversion+of+5-methylcytosine+to+5-hydroxymethylcytosine+in+mammalian+DNA+by+MLL+partner+TET1.">Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1.</a> Science. 324 (5929), 930-935 (2009).</li><li>Wu, H., Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+and+functions+of+Tet+protein-mediated+5-methylcytosine+oxidation.">Mechanisms and functions of Tet protein-mediated 5-methylcytosine oxidation.</a> Genes Dev. 25 (23), 2436-2452 (2011).</li><li>Inoue, A., Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication-dependent+loss+of+5-hydroxymethylcytosine+in+mouse+preimplantation+embryos.">Replication-dependent loss of 5-hydroxymethylcytosine in mouse preimplantation embryos.</a> Science. 334 (6053), 194 (2011).</li><li>He, Y. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tet-mediated+formation+of+5-carboxylcytosine+and+its+excision+by+TDG+in+mammalian+DNA.">Tet-mediated formation of 5-carboxylcytosine and its excision by TDG in mammalian DNA.</a> Science. 333 (6047), 1303-1307 (2011).</li><li>Maiti, A., Drohat, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thymine+DNA+glycosylase+can+rapidly+excise+5-formylcytosine+and+5-carboxylcytosine:+Potential+implications+for+active+demethylation+of+CpG+sites.">Thymine DNA glycosylase can rapidly excise 5-formylcytosine and 5-carboxylcytosine: Potential implications for active demethylation of CpG sites.</a> J. Biol. Chem. 286 (41), 35334-35338 (2011).</li><li>Habibi, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-genome+bisulfite+sequencing+of+two+distinct+interconvertible+DNA+methylomes+of+mouse+embryonic+stem+cells.">Whole-genome bisulfite sequencing of two distinct interconvertible DNA methylomes of mouse embryonic stem cells.</a> Cell Stem Cell. 13 (3), 360-369 (2013).</li><li>von Meyenn, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impairment+of+DNA+methylation+maintenance+is+the+main+cause+of+global+demethylation+in+naive+embryonic+stem+cells.">Impairment of DNA methylation maintenance is the main cause of global demethylation in naive embryonic stem cells.</a> Mol. Cell. 62, 848-861 (2016).</li><li>Li, C., Liu, B., Zhong, S., Wang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MEK+inhibitor+PD0325901+and+vitamin+C+synergistically+induce+hypomethylation+of+mouse+embryonic+stem+cells.">MEK inhibitor PD0325901 and vitamin C synergistically induce hypomethylation of mouse embryonic stem cells.</a> Oncotarget. 7 (26), 39730-39739 (2016).</li><li>Hackett, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergistic+mechanisms+of+DNA+demethylation+during+transition+to+ground-state+pluripotency.">Synergistic mechanisms of DNA demethylation during transition to ground-state pluripotency.</a> Stem Cell Reports. 1 (6), 518-531 (2013).</li><li>Nakaki, F., Saitou, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PRDM14:+a+unique+regulator+for+pluripotency+and+epigenetic+reprogramming.">PRDM14: a unique regulator for pluripotency and epigenetic reprogramming.</a> Trends Biochem. 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The authors disclose no potential conflicts of interest.

In the work, we demonstrated a novel method of combining Vc and PD0325901 to sustain mouse ES cells at an undifferentiated and hypomethylated state, which was achieved by a synergistic action of promoting DNA demethylation by Vc and suppressing de novo DNA methylation by PD0325901. Moreover, mouse ES cells showed great morphology under the Vc/PD0325901 culture system.
To better sustain the state of mouse ES cells in the Vc/PD0325901 culture system, there are some critical steps. First...

ES cells are originated from the ICM of a blastocyst1. The cells are in a pluripotency state and can form all somatic lineages and germline cells2. Establishment of ES cells provides the opportunity to investigate the development processes in vitro and can generate cells of medical relevance for regenerative medicine based on their pluripotency3.
Two groups seminally established the mouse ES cell lines in 1981 and when cells derived from the early mouse embryo were cultured in fetal bovine serum (FBS)-containing medium with mouse embryonic fibroblasts (MEFs) as the feeder layer1,4. MEFs were inactivated mitotically and were pre-grown in dishes prior to culturing ES cells. MEFs provide support for mouse ES cell attachment and produce growth factors to promote propagation and repress the differentiation5, while FBS offers essential trophic factors and hormones for cell proliferation. Subsequent studies indicated that LIF produced by feeder cells was the key cytokine for self-renewal and maintenance of pluripotency in mouse ES cells, and the addition of LIF into the medium could substitute for feeder cells6. Currently, the sustainment of mouse ES cells in FBS/LIF medium on feeders is still the standard method adopted by many researchers. However, some problems arise with this classical culture approach. Firstly, feeder cells secrete excess and uncontrolled factors and may cause pathogenic contamination7. To avoid this interference, coating the surface of dishes with gelatin and the addition of LIF in serum-containing medium are alternative methods for maintaining mouse ES cells without feeder-layer cells. Additionally, mouse ES cells grown under serum/LIF conditions exhibit morphological heterogeneity in cell populations and even in the expression level of pluripotency-related factors8. Recent studies suggest that under serum/LIF conditions, the pluripotency-related core transcription factors (including SOX2, Nanog, and OCT4) can sustain the pluripotency through LIF and WNT signaling; however, notably, they also activate a fibroblast growth factor (FGF) signal to trigger differentiation8. Due to the ambivalent dual action of the core transcription factors, mouse ES cells cultured in serum present heterogeneity, consisting of two interchangeable populations, one similar to ICM and another resembling the primed epiblast state8. Moreover, mouse ES cells in serum often exhibit global hypermethylation9, whereas ICM and PGCs are in a global hypomethylated state, which is closely linked with their pluripotency9,10.
There is a considerable demand to develop new methods for culturing mouse ES cells. Several improved protocols have been established since 200311 but there continues to be some limitations and shortcomings7. Since 2008, the combined utilization of two small-molecule kinase inhibitors, PD0325901 (the inhibitor of the mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated kinase (ERK) (MEK)) and CHIR99021 (the inhibitor of glycogen synthase kinase 3 (GSK3)), in N2B27 medium with LIF and without serum has opened new perspectives for culturing mouse ES cells12. This new defined medium is characterized by the use of two inhibitors (2i). Mouse ES cells cultured in 2i/LIF medium are more homogeneous in cell populations and the expression of pluripotency factors. In addition, 2i/LIF-cultured mouse ES cells exhibit DNA hypomethylation globally, which is closer to ICM-like cells9,13. Even so, 2i culture has its disadvantages. PD0325901 and CHIR99021 are insoluble in water and generally are dissolved in dimethyl sulfoxide (DMSO)-based stock solution to add them in culture medium. Studies have showed that long-term and low-dose exposure of cells to DMSO can lead to cytotoxicity14.
Here, we utilized two small-molecule compounds and developed a new culture method of mouse ES cells. The novel culture method combines Vc and MEK inhibitor PD0325901 to promote DNA hypomethylation rapidly and effectively to a comparable level of PGC, named as the Vc/PD0325901 culturing protocol. Mouse ES cells in Vc/PD0325901-added serum-containing medium exhibit homogeneity in morphology and are sustained in a ground state. Compared to 2i culture, mouse ES cells cultured under Vc/PD0325901 conditions exhibit faster kinetics of DNA demethylation and can reach the hypomethylation level comparable to that of PGC. In addition, the use of a single inhibitor (PD0325901) decreases the input amount of DMSO into medium in comparison to that used in 2i (PD0325901/CHIR99021) and reduces the damage to cells.

<table border="0" cellpadding="0" cellspacing="0" width="790">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">fetal bovine serum Australia source</td>
			<td style="width:205px;">Corning</td>
			<td style="width:119px;">35-076-CV</td>
			<td style="width:251px;">Component of mouse ES cell medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM/high glucose</td>
			<td style="width:205px;">Hyclone</td>
			<td style="width:119px;">SH30022</td>
			<td>Component of mouse ES cell medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LIF</td>
			<td>Millipore</td>
			<td style="width:119px;">ESG1107</td>
			<td>Component of mouse ES cell medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">non-essential amino acids (NEAA)</td>
			<td style="width:205px;">Gibco</td>
			<td style="width:119px;">11140050</td>
			<td>Component of mouse ES cell medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sodium pyruvate</td>
			<td style="width:205px;">Gibco</td>
			<td style="width:119px;">11360070</td>
			<td>Component of mouse ES cell medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-glutamine</td>
			<td style="width:205px;">Gibco</td>
			<td style="width:119px;">25030081</td>
			<td>Component of mouse ES cell medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">penicillin streptomycin solution</td>
			<td style="width:205px;">Gibco</td>
			<td style="width:119px;">15140122</td>
			<td>Component of mouse ES cell medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS</td>
			<td style="width:205px;">Sigma</td>
			<td style="width:119px;">P5493</td>
			<td>Cells rinse</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">trypsin</td>
			<td style="width:205px;">Hyclone</td>
			<td style="width:119px;">SH30042</td>
			<td>Cell dissociation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">gelatin</td>
			<td style="width:205px;">Sigma</td>
			<td style="width:119px;">48722</td>
			<td>Dishes coating</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PD0325901</td>
			<td>Stemolecule</td>
			<td>04-0006-10</td>
			<td>small-molecule chemical for cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CHIR99021</td>
			<td>Stemolecule</td>
			<td>04-0004</td>
			<td>small-molecule chemical for cell culture</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">vitamin C</td>
			<td style="width:205px;">Sinopharm Chemical Reagent Co.,Ltd&#160;</td>
			<td style="width:119px;">XW00508171</td>
			<td>small-molecule chemical for cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultra Bioscience water purification systemr</td>
			<td style="width:205px;">Purelab</td>
			<td style="width:119px;">Ultra</td>
			<td>Ultra water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMSO</td>
			<td style="width:205px;">Sigma</td>
			<td style="width:119px;">D8418</td>
			<td>Cell freezing medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">centrifuger</td>
			<td style="width:205px;">Eppendorf</td>
			<td style="width:119px;">5427R</td>
			<td>DNA and protein extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">protease inhibitor cocktail</td>
			<td style="width:205px;">Sigma</td>
			<td style="width:119px;">P8340</td>
			<td>Component of RIPA buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">PMSF Protease Inhibitor</td>
			<td style="width:205px;">ThermoFisher Scientific</td>
			<td style="width:119px;">36978</td>
			<td>Component of RIPA buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DTT</td>
			<td style="width:205px;">Sigma</td>
			<td style="width:119px;">43815</td>
			<td>Component of RIPA buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EGTA</td>
			<td style="width:205px;">Sigma</td>
			<td style="width:119px;">E3889</td>
			<td>Component of RIPA buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Genomic DNA Purification Kit&#160;</td>
			<td style="width:205px;">Promega</td>
			<td style="width:119px;">A1125</td>
			<td>Genomic DNA extraction</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">NanoDrop 2000</td>
			<td style="width:205px;">ThermoFisher Scientific</td>
			<td style="width:119px;">NanoDrop 2000</td>
			<td>Determination of DNA concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">calf intestinal phosphatase</td>
			<td>New England Biolabs</td>
			<td style="width:119px;">M0290</td>
			<td>DNA digestion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNase I</td>
			<td>New England Biolabs</td>
			<td style="width:119px;">M0303</td>
			<td>DNA digestion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">snake venom phosphodiesterase I</td>
			<td style="width:205px;">Sigma</td>
			<td style="width:119px;">P4506</td>
			<td>DNA digestion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nanosep 3K Omega</td>
			<td>Pall</td>
			<td style="width:119px;">OD003C35</td>
			<td>filtration of digested DNA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1290 UHPLC system</td>
			<td style="width:205px;">Agilent&#160;</td>
			<td style="width:119px;">1290</td>
			<td>UHPLC separation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">G6410B triple quadrupole mass spectrometer</td>
			<td style="width:205px;">Agilent&#160;</td>
			<td style="width:119px;">G6410B</td>
			<td>MS/MS analysis</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Zorbax Eclipse Plus C18 column</td>
			<td style="width:205px;">Agilent&#160;</td>
			<td style="width:119px;">Zorbax Eclipse Plus</td>
			<td>colume for UHPLC separation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">5-methylcytosine</td>
			<td>Solarbio Life Sciences</td>
			<td style="width:119px;">SM8900&#160;</td>
			<td>standard 5mC</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">5-hydroxymethylcytosine (standard)</td>
			<td>Toronto Research Chemicals</td>
			<td style="width:119px;">M295900</td>
			<td>standard 5hmC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Prdm14 antibody</td>
			<td>bioworld</td>
			<td>BS7634</td>
			<td>Western blot analysis-primary antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dnmt3a antibody</td>
			<td>bioworld</td>
			<td>BS6587</td>
			<td>Western blot analysis-primary antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dnmt3b antibody</td>
			<td>abcam</td>
			<td>ab13604</td>
			<td>Western blot analysis-primary antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dnmt3l antibody</td>
			<td>abcam</td>
			<td>ab3493</td>
			<td>Western blot analysis-primary antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dnmt1 antibody</td>
			<td>abcam</td>
			<td>ab13537</td>
			<td>Western blot analysis-primary antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#946;-tubulin antibody</td>
			<td>bioworld</td>
			<td>BS1482MH</td>
			<td>Western blot analysis-primary antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">goat anti-rabbit IgG</td>
			<td>abcam</td>
			<td style="width:119px;">ab6721</td>
			<td>Western blot analysis-secondary antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">goat anti-mouse IgG</td>
			<td>abcam</td>
			<td style="width:119px;">ab6789</td>
			<td>Western blot analysis-secondary antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trizol</td>
			<td style="width:205px;">Invitrogen</td>
			<td>15596-026</td>
			<td>RNA extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">reverse transcription system</td>
			<td style="width:205px;">Promega</td>
			<td style="width:119px;">A3500</td>
			<td>RNA reverse transcription</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GoTaq qPCR Master Mix</td>
			<td style="width:205px;">Promega</td>
			<td style="width:119px;">A6001</td>
			<td>RT-PCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">StemTAG alkaline phosphatase staining and activity assay kit</td>
			<td style="width:205px;">Cells Biolabs, Inc.</td>
			<td>CBA-302</td>
			<td>&#160;alkaline phosphatase staining analysis</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">mouse ES cells: WT, 129 SvEv</td>
			<td style="width:205px;">Provided by Professor Guoliang Xu (Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China)</td>
			<td style="width:119px;"></td>
			<td>cell strain of mouse ES cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">microscope</td>
			<td style="width:205px;">Zeiss</td>
			<td style="width:119px;">LSM510</td>
			<td>cells observation</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:216px;">GraphPad Prism 5.0</td>
			<td style="width:205px;">GraphPad Prism Software Inc.</td>
			<td style="width:119px;">5.0</td>
			<td>statistical analysis</td>
		</tr>
	</tbody>
</table>

an alternative culture method to maintain genomic hypomethylation of mouse embryonic stem cells using mek inhibitor pd0325901 and vitamin c

Embryonic stem (ES) cells have the potential to differentiate into any of the three germ layers (endoderm, mesoderm, or ectoderm), and can generate many lineages for regenerative medicine. ES cell culture in vitro has long been the subject of widespread concerns. Classically, mouse ES cells are maintained in serum and leukemia inhibitory factor (LIF)-containing medium. However, under serum/LIF conditions, cells show heterogeneity in morphology and the expression profile of pluripotency-related genes, and are mostly in a metastable state. Moreover, cultured ES cells exhibit global hypermethylation, but na&#239;ve ES cells of the inner cell mass (ICM) and primordial germ cells (PGCs) are in a state of global hypomethylation. The hypomethylated state of ICM and PGCs is closely associated with their pluripotency. To improve mouse ES cell culture methods, we have recently developed a new method based on the selectively combined utilization of two small-molecule compounds to maintain the DNA hypomethylated and pluripotent state. Here, we present that the co-treatment of vitamin C (Vc) and PD0325901 can erase about 90% of 5-methylcytosine (5mC) at 5 days in mouse ES cells. The generated 5mC content is comparable to that in PGCs. The mechanistic investigation shows that PD0325901 up-regulates Prdm14 expression to suppress Dnmt3b (de novo DNA methyltransferase) and Dnmt3l (the cofactor of Dnmt3b), by reducing de novo 5mC synthesis. Vc facilitates the conversion of 5mC to 5-hydroxymethylcytosine (5hmC) catalyzed mainly by Tet1 and Tet2, indicating the involvement of both passive and active DNA demethylations. Moreover, under Vc/PD0325901 conditions, mouse ES cells show homogeneous morphology and pluripotent state. Collectively, we propose a novel and chemical-synergy culture method for achieving DNA hypomethylation and maintenance of pluripotency in mouse ES cells. The small-molecule chemical-dependent method overcomes the major shortcomings of serum culture, and holds promise to generate homogeneous ES cells for further clinical applications and researches.

Vc/PD0325901 synergistically induced global erasure of mouse ES cells. Mouse ES cells in serum exhibit DNA hypermethylation, while pluripotent ICM cells and PGCs show global erasure of DNA methylation and the hypomethylated state is closely associated with their pluripotency9,10.
Previously, we and others found that Vc may enhance Tet-mediated 5mC demethylation<sup class...

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An Alternative Culture Method to Maintain Genomic Hypomethylation of Mouse Embryonic Stem Cells Using MEK Inhibitor PD0325901 and Vitamin C

We described in detail two chemical-based protocols for culturing mouse embryonic stem cells. This new method utilizes synergistic mechanisms of promoting Tet-mediated oxidation (by vitamin C) and repressing de novo synthesis of 5-methylcytosine (by PD0325901) to maintain DNA hypomethylation and pluripotency of mouse embryonic stem cells.

Embryonic stem (ES) cells have the potential to differentiate into any of the three germ layers (endoderm, mesoderm, or ectoderm), and can generate many ...

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Research

JoVE Journal

Biochemistry

6.6K Views.  Chinese Academy of Sciences. We described in detail two chemical-based protocols for culturing mouse embryonic stem cells. This new method utilizes synergistic mechanisms of promoting Tet-mediated oxidation (by vitamin C) and repressing de novo synthesis of 5-methylcytosine (by PD0325901) to maintain DNA hypomethylation and pluripotency of mouse embryonic stem cells.

Video: An Alternative Culture Method to Maintain Genomic Hypomethylation of Mouse Embryonic Stem Cells Using MEK Inhibitor PD0325901 and Vitamin C

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For a long time, the gene expression studies were dominated by research on the transcriptional level. However, the steady-state levels of mRNAs do not correlate well with protein production, and the translatability of mRNAs varies greatly depending on the conditions. In some organisms, like the parasite Leishmania, the protein expression is regulated mostly at the translational level. Recent studies demonstrated that protein translation dysregulation is associated with cancer, metabolic, neurodegenerative and other human diseases. Polysome profiling is a powerful method to study protein translation regulation. It allows to measure the translational status of individual mRNAs or examine translation on a genome-wide scale. The basis of this technique is the separation of polysomes, ribosomes, their subunits and free mRNAs during centrifugation of a cytoplasmic lysate through a sucrose gradient. Here, we present a universal polysome profiling protocol used on three different models - parasite Leishmania major, cultured human cells and animal tissues. Leishmania cells freely grow in suspension and cultured human cells grow in adherent monolayer, while mouse testis represents an animal tissue sample. Thus, the technique is adapted to all of these sources. The protocol for the analysis of polysomal fractions includes detection of individual mRNA levels by RT-qPCR, proteins by Western blot and analysis of ribosomal RNAs by electrophoresis. The method can be further extended by examination of mRNAs association with the ribosome on a transcriptome level by deep RNA-seq and analysis of ribosome-associated proteins by mass spectroscopy of the fractions. The method can be easily adjusted to other biological models.

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

The overall goal of polysome profiling technique is analysis of translational activity of individual mRNAs or transcriptome mRNAs during protein synthesis. The method is important for studies of protein synthesis regulation, translation activation and repression in health and multiple human diseases.

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For ...

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic properties of the absorbed/immobilized mass on sensor surfaces, allowing the measurements of the interaction of proteins with solid-supported surfaces, such as lipid bilayers, in real-time and with a high sensitivity. Annexins are a highly conserved group of phospholipid-binding proteins that interact reversibly with the negatively charged headgroups via the coordination of calcium ions. Here, we describe a protocol that was employed to quantitatively analyze the binding of annexin A2 (AnxA2) to planar lipid bilayers prepared on the surface of a quartz sensor. This protocol is optimized to obtain robust and reproducible data and includes a detailed step-by-step description. The method can be applied to other membrane-binding proteins and bilayer compositions.

Here, we present an experimental protocol that can be employed to determine the binding affinities and mode of interaction of label-free phospholipid-binding protein annexin A2 with immobilized solid-supported bilayers (SLB) by simultaneously measuring the mass uptake and the viscoelastic properties of the protein annexin A2.

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

DNA Electrophoresis Using Thiazole Orange Instead of Ethidium Bromide or Alternative Dyes

DNA gel electrophoresis using agarose is a common tool in molecular biology laboratories, allowing separation of DNA fragments by size. After separation, DNA is visualized by staining. This article demonstrates how to use thiazole orange to stain DNA. Thiazole orange compares favorably to common staining methods, in that it is sensitive, inexpensive, excitable with UV or blue light (to prevent sample damage), and safer than ethidium bromide. Labs already equipped to run DNA electrophoresis experiments using ethidium bromide can generally switch dyes with no additional changes to existing protocols, using UV light for detection. Blue-light detection to avoid sample damage can additionally be achieved with a blue-light source and emission filter. Labs already equipped for blue-light detection can simply switch dyes with no additional changes to existing protocols.

Here, we present a protocol to use thiazole orange for the detection of DNA in gel electrophoresis experiments. The use of thiazole orange allows elimination of ethidium bromide, and fluorescence detection can be achieved with either UV or blue light.

DNA gel electrophoresis using agarose is a common tool in molecular biology laboratories, allowing separation of DNA fragments by size. After separation, ...

DNA Sequence Recognition by DNA Primase Using High-Throughput Primase Profiling

DNA primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. The binding of prokaryotic DnaG-like primases to DNA occurs at a specific trinucleotide recognition sequence. It is a pivotal step in the formation of Okazaki fragments. Conventional biochemical tools that are used to determine the DNA recognition sequence of DNA primase provide only limited information. Using a high-throughput microarray-based binding assay and consecutive biochemical analyses, it has been shown that 1) the specific binding context (flanking sequences of the recognition site) influences the binding strength of the DNA primase to its template DNA, and 2) stronger binding of primase to the DNA yields longer RNA primers, indicating higher processivity of the enzyme. This method combines PBM and primase activity assay and is designated as high-throughput primase profiling (HTPP), and it allows characterization of specific sequence recognition by DNA primase in unprecedented time and scalability.&#160;

Protein binding microarray (PBM) experiments combined with biochemical assays link the binding and catalytic properties of DNA primase, an enzyme that synthesizes RNA primers on template DNA. This method, designated as high-throughput primase profiling (HTPP), can be used to reveal DNA-binding patterns of a variety of enzymes.

DNA primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. ...

Kinase Inhibitor Screening In Self-assembled Human Protein Microarrays

The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with clinical implications. Several methodologies have been reported to accomplish such screening. However, each has its own limitations (e.g., the screening of only ATP analogues, restriction to using purified kinase domains, significant costs associated with testing more than a few kinases at a time, and lack of flexibility in screening protein kinases with novel mutations). Here, a new protocol that overcomes some of these limitations and can be used for the unbiased screening of kinase inhibitors is presented. A strength of this method is its ability to compare the activity of kinase inhibitors across multiple proteins, either between different kinases or different variants of the same kinase. Self-assembled protein microarrays generated through the expression of protein kinases by a human-based in vitro transcription and translation system (IVTT) are employed. The proteins displayed on the microarray are active, allowing for measurement of the effects of kinase inhibitors. The following procedure describes the protocol steps in detail, from the microarray generation and screening to the data analysis.

A detailed protocol for the generation of self-assembled human protein microarrays for the screening of kinase inhibitors is presented.

The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with ...

Using an Extracellular Flux Analyzer to Measure Changes in Glycolysis and Oxidative Phosphorylation during Mouse Sperm Capacitation

Mammalian sperm acquire fertilization capacity in the female reproductive tract in a process known as capacitation. Capacitation-associated processes require energy. There remains an ongoing debate about the sources generating the ATP which fuels sperm progressive motility, capacitation, hyperactivation, and acrosome reaction. Here, we describe the application of an extracellular flux analyzer as a tool to analyze changes in energy metabolism during mouse sperm capacitation. Using H+- and O2- sensitive fluorophores, this method allows monitoring glycolysis and oxidative phosphorylation in real-time in non-capacitated versus capacitating sperm. Using this assay in the presence of different energy substrates and/or pharmacological activators and/or inhibitors can provide important insights into the contribution of different metabolic pathways and the intersection between signaling cascades and metabolism during sperm capacitation.

We describe the application of an extracellular flux analyzer to monitor real-time changes in glycolysis and oxidative phosphorylation during mouse sperm capacitation.

Mammalian sperm acquire fertilization capacity in the female reproductive tract in a process known as capacitation. Capacitation-associated processes ...

Characterization of Amyloid Structures in Aging C. Elegans Using Fluorescence Lifetime Imaging

Amyloid fibrils are associated with a number of neurodegenerative diseases such as Huntington&#39;s, Parkinson&#39;s, or Alzheimer&#39;s disease. These amyloid fibrils can sequester endogenous metastable proteins as well as components of the proteostasis network (PN) and thereby exacerbate protein misfolding in the cell. There are a limited number of tools available to assess the aggregation process of amyloid proteins within an animal. We present a protocol for fluorescence lifetime microscopy (FLIM) that allows monitoring as well as quantification of the amyloid fibrilization in specific cells, such as neurons, in a noninvasive manner and with the progression of aging and upon perturbation of the PN. FLIM is independent of the expression levels of the fluorophore and enables an analysis of the aggregation process without any further staining or bleaching. Fluorophores are quenched when they are in close vicinity of amyloid structures, which results in a decrease of the fluorescence lifetime. The quenching directly correlates with the aggregation of the amyloid protein. FLIM is a versatile technique that can be applied to compare the fibrilization process of different amyloid proteins, environmental stimuli, or genetic backgrounds in vivo in a non-invasive manner.

Characterization of Amyloid Structures in Aging C. Elegans Using Fluorescence Lifetime Imaging

Fluorescence lifetime imaging monitors, quantifies and distinguishes the aggregation tendencies of proteins in living, aging, and stressed C. elegans disease models.

Amyloid fibrils are associated with a number of neurodegenerative diseases such as Huntington&#39;s, Parkinson&#39;s, or Alzheimer&#39;s disease. These ...

Improved UPLC-UV Method for the Quantification of Vitamin C in Lettuce Varieties (Lactuca sativa L.) and Crop Wild Relatives (Lactuca spp.)

Vitamins, especially vitamin C, are important micronutrients found in fruits and vegetables. Vitamin C is also a major contributor to their antioxidant capacity. Lettuce is one of the most popular vegetables among consumers worldwide. An accurate protocol to measure vitamin C content in lettuce and other related species is crucial. We describe here a method using the ultra-high-performance liquid chromatography-ultraviolet (UPLC-UV) technique, in which sample preparation, vitamin extraction and chromatography conditions were optimized.
Samples were collected to represent the entire plant, frozen at -80 &#176;C and lyophilized to prevent undesirable oxidation and make their manipulation easier. The extraction of vitamin C was carried out in acidic media, which also contributed to its stability. As vitamin C can be present in two different interconvertible forms, ascorbic acid (AA) and dehydroascorbic acid (DHAA), both compounds should be measured for accurate quantification. The DHAA was quantified indirectly after its reduction to AA because AA shows a higher absorptivity than DHAA in the UV range of the spectrum. From the same extract, two measurements were carried out, one before and one after that reduction reaction. In the first case, we were quantifying the AA content, and in the second one, we quantified the sum of AA and DHAA (TAA: total ascorbic acid) in the form of AA. Then, DHAA quantity was indirectly obtained by subtracting AA coming from the first measurement from TAA. They were determined by UPLC-UV, using a commercial AA standard to build a calibration curve and optimizing the chromatographic procedure, to obtain AA peaks that were completely resolved in a short time. This protocol could be easily extrapolated to any other plant material with slight or no changes. Its accuracy revealed statistically significant differences otherwise unperceived. Other strengths and limitations are discussed more in depth in the manuscript.

Improved UPLC-UV Method for the Quantification of Vitamin C in Lettuce Varieties Lactuca sativa L. and Crop Wild Relatives Lactuca spp.

We present a fast and reliable method to quantify vitamin C in Lactuca spp. using UPLC-UV, potentially transferable to other plants. The key steps are the sample preparation and vitamin C extraction under stable conditions, the reduction of dehydroascorbic acid to ascorbic acid and the optimization of the chromatographic procedure.

Vitamins, especially vitamin C, are important micronutrients found in fruits and vegetables. Vitamin C is also a major contributor to their antioxidant ...

Synthesis of Masarimycin, a Small Molecule Inhibitor of Gram-Positive Bacterial Growth

Peptidoglycan (PG) in the cell wall of bacteria is a unique macromolecular structure that confers shape, and protection from the surrounding environment. Central to understanding cell growth and division is the knowledge of how PG degradation influences biosynthesis and cell wall assembly. Recently, the metabolic labeling of PG through the introduction of modified sugars or amino acids has been reported. While chemical interrogation of biosynthetic steps with small molecule inhibitors is possible, chemical biology tools to study PG degradation by autolysins are underdeveloped. Bacterial autolysins are a broad class of enzymes that are involved in the tightly coordinated degradation of PG. Here, a detailed protocol is presented for preparing a small molecule probe, masarimycin, which is an inhibitor of N-acetylglucosaminidase LytG in Bacillus subtilis, and cell wall metabolism in Streptococcus pneumoniae. Preparation of the inhibitor via microwave-assisted and classical organic synthesis is provided. Its applicability as a tool to study Gram-positive physiology in biological assays is presented.

A detailed protocol is presented for preparing the bacteriostatic diamide masarimycin, a small molecule probe that inhibits the growth of Bacillus subtilis and Streptococcus pneumoniae by targeting cell wall degradation. Its application as a chemical probe is demonstrated in synergy/antagonism assays and morphological studies with B. subtilis and S. pneumoniae.

Peptidoglycan (PG) in the cell wall of bacteria is a unique macromolecular structure that confers shape, and protection from the surrounding environment. ...

Deciphering Molecular Mechanism of Histone Assembly by DNA Curtain Technique

Chromatin is a higher-order structure that packages eukaryotic DNA. Chromatin undergoes dynamic alterations according to the cell cycle phase and in response to environmental stimuli. These changes are essential for genomic integrity, epigenetic regulation, and DNA metabolic reactions such as replication, transcription, and repair. Chromatin assembly is crucial for chromatin dynamics and is catalyzed by histone chaperones. Despite extensive studies, the mechanisms by which histone chaperones enable chromatin assembly remains elusive. Moreover, the global features of nucleosomes organized by histone chaperones are poorly understood. To address these problems, this work describes a unique single-molecule imaging technique named DNA curtain, which facilitates the investigation of the molecular details of nucleosome assembly by histone chaperones. DNA curtain is a hybrid technique that combines lipid fluidity, microfluidics, and total internal reflection fluorescence microscopy (TIRFM) to provide a universal platform for real-time imaging of diverse protein-DNA interactions.Using DNA curtain, the histone chaperone function of Abo1, the Schizosaccharomyces pombe bromodomain-containing AAA+ ATPase, is investigated, and the molecular mechanism underlying histone assembly of Abo1 is revealed. DNA curtain provides a unique approach for studying chromatin dynamics.

DNA curtain, a high-throughput single-molecule imaging technique, provides a platform for real-time visualization of diverse protein-DNA interactions. The present protocol utilizes the DNA curtain technique to investigate the biological role and molecular mechanism of Abo1, a Schizosaccharomyces pombe bromodomain-containing AAA+ ATPase.

Chromatin is a higher-order structure that packages eukaryotic DNA. Chromatin undergoes dynamic alterations according to the cell cycle phase and in ...