This study was supported by grants from the Science and Technology Commission of Shanghai (Grant No. 14411961800) and National Natural Science Foundation of China (Grant No. 81300805).

1. Detection of Endogenous Rb SUMOylation at the Early G1 Phase
<ol>
	<li>Cell culture and cell cycle synchronization.
	<ol>
		<li>Maintain HEK293 cells in growth medium containing Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 1% Pen-Strep and 10% fetal bovine serum (FBS) at 37 &#176;C and 5% CO2 in an incubator.</li>
		<li>Synchronize the HEK293 cells at the G0 phase.
		<ol>
			<li>Count the HEK293 cells using a hemocytometer and seed ~1.5 x&#160;107 cells in a 15...

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+retinoblastoma+protein+and+cell+cycle+control.">The retinoblastoma protein and cell cycle control.</a> Cell. 81 (3), 323-330 (1995).</li><li>Giacinti, C., Giordano, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RB+and+cell+cycle+progression.">RB and cell cycle progression.</a> Oncogene. 25 (38), 5220-5227 (2006).</li><li>Burkhart, D. L., Sage, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+mechanisms+of+tumour+suppression+by+the+retinoblastoma+gene.">Cellular mechanisms of tumour suppression by the retinoblastoma gene.</a> Nat Rev Cancer. 8 (9), 671-682 (2008).</li><li>Ferreira, R., Naguibneva, I., Pritchard, L. 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This paper describes a protocol to detect and analyze the endogenous SUMOylation of Rb in human cells. As this method is specifically focused on the endogenous Rb protein without any alternation of global SUMO-related signal, it is an important tool for investigating Rb-SUMO modification under completely natural physiologic circumstances.
To achieve this aim, it is important to keep in mind that: 1) although SUMO comprises four isoforms (SUMO1-4, each encoded by different genes)in comparison t...

The accurate control of cell cycle progression in eukaryotic cells is based on a tight regulatory network, which ensures that particular events take place in an ordered manner1,2. One of the key players in this network is the retinoblastoma (Rb) protein, the first cloned tumor suppressor1,3. The Rb protein is thought to be a negative regulator of cell cycle progression, especially for the G0/G1 to S phase transition, and tumor growth4,5. Failure of Rb function either directly leads to the most common intraocular malignancy in children, retinoblastoma, or contributes to the development of many other types of cancer5. Moreover, Rb is involved in many cellular pathways including cell differentiation, chromatin remodeling, and mitochondria-mediated apoptosis3,6,7.
Post-translational modifications play a pivotal role in the regulation of RB function8,9. Phosphorylation is one such modification, and it usually leads to Rb inactivation. In quiescent G0 cells, Rb is active with a low phosphorylation level. As cells progress through G0/G1 phase, Rb is sequentially hyper-phosphorylated by a series of cyclin-dependent protein kinases (CDKs) and cyclins, such as cyclin E/CDK2 and cyclin D/CDK4/6, which inactivate Rb and eliminate its ability to repress cell-cycle related gene expression4,10. Rb could also be modified by small ubiquitin-related modifier (SUMO)11,12,13.
SUMO is a ubiquitin-like protein that is covalently attached to a variety of target proteins. It is crucial for diverse cellular processes, including cell cycle regulation, transcription, protein cellular localization and degradation, transport, and DNA repair14,15,16,17,18. The SUMO conjugation pathway consists of the dimeric SUMO E1 activating enzyme SAE1/UBA2, the single E2 conjugating enzyme Ubc9, multiple E3 ligases, and SUMO-specific proteases. Generally, nascent SUMO proteins must be proteolytically processed to generate the mature form. The mature SUMO is activated by the E1 heterodimer and then transferred to the E2 enzyme Ubc9. Finally, the C-terminal glycine of SUMO is covalently conjugated to the target lysine of a substrate, and this process is usually facilitated by E3 ligases. The SUMO protein can be removed from the modified substrate by specific proteases. A previous study by the authors of this paper revealed that SUMOylation of Rb increases its binding to CDK2, leading to hyper-phosphorylation at the early G1 phase, a process which is necessary for cell cycle progression13. We also demonstrated that the loss of Rb SUMOylation causes a decreased cell proliferation. Moreover, it was recently demonstrated that the SUMOylation of Rb protects the Rb protein from proteasomal turnover, thus increasing the level of Rb protein in cells19. Therefore, SUMOylation plays an important role in Rb function in various cellular processes. To further study the functional consequence and physiological relevance of Rb SUMOylation, it is important to develop an effective method to analyze the SUMO status of Rb in human cells or patient tissues.
SUMOylation is a reversible, highly dynamic process. Thus, it is usually difficult to detect the SUMO-modified proteins under completely endogenous conditions. This paper presents a method to detect endogenous Rb SUMOylation. Furthermore, it shows how to detect exogenous Rb SUMOylation of both wild-type Rb and its SUMO-deficient mutation11. In particular, Jacobs et al. described a method to increase the SUMO modification of a given substrate specifically by Ubc9 fusion-directed SUMOylation (UFDS)20. Based on this method, this protocol describes how to analyze the forced SUMOylation of Rb and its functional consequences. Given that hundreds of SUMO substrates have been described previously and more putative SUMO substrates have been identified from many proteomic-based assays, this protocol can be applied to analyze the SUMO-modification of these proteins in human cells.

<table style="border-collapse:collapse;width:695pt" width="927">
	<colgroup>
		<col style="width:275pt" width="366" />
		<col style="width:206pt" width="275" />
		<col style="width:89pt" width="119" />
		<col style="width:125pt" width="167" />
	</colgroup>
	<tbody>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:275pt;height:15.75pt" width="366">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td class="xl65" style="width:206pt" width="275">Thermo Fisher Scientific</td>
			<td class="xl70" style="width:89pt" width="119">11995065</td>
			<td class="xl68" style="width:125pt" width="167"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl68" height="21" style="height:15.75pt">Opti-MEM&#160;</td>
			<td class="xl65" style="width:206pt" width="275">Thermo Fisher Scientific</td>
			<td class="xl70" style="width:89pt" width="119">31985070</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:275pt;height:15.75pt" width="366">Fetal Bovine Serum (FBS)</td>
			<td class="xl65" style="width:206pt" width="275">Thermo Fisher Scientific</td>
			<td class="xl70" style="width:89pt" width="119">26140079</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:275pt;height:15.75pt" width="366">Penicillin-Streptomycin</td>
			<td class="xl65" style="width:206pt" width="275">Thermo Fisher Scientific</td>
			<td class="xl70" style="width:89pt" width="119">15140122</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:275pt;height:15.75pt" width="366">Phosphate-buffered Saline (PBS)</td>
			<td class="xl65" style="width:206pt" width="275">Thermo Fisher Scientific</td>
			<td class="xl70" style="width:89pt" width="119">10010023</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:275pt;height:15.75pt" width="366">Trypsin-EDTA</td>
			<td class="xl65" style="width:206pt" width="275">Thermo Fisher Scientific</td>
			<td class="xl70" style="width:89pt" width="119">25200056</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:275pt;height:15.75pt" width="366">Thymidine</td>
			<td class="xl65" style="width:206pt" width="275">Sigma</td>
			<td class="xl70" style="width:89pt" width="119">T9250</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:275pt;height:15.75pt" width="366">Nocodazole</td>
			<td class="xl65" style="width:206pt" width="275">Sigma</td>
			<td class="xl70" style="width:89pt" width="119">M1404</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl68" height="21" style="height:15.75pt">propidium iodide</td>
			<td class="xl65" style="width:206pt" width="275">Thermo Fisher Scientific</td>
			<td class="xl70" style="width:89pt" width="119">P3566</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:275pt;height:15.75pt" width="366">Triton X-100&#160;</td>
			<td class="xl65" style="width:206pt" width="275">AMRESCO</td>
			<td class="xl70" style="width:89pt" width="119">694</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:275pt;height:15.75pt" width="366">RNase A&#160;</td>
			<td class="xl65" style="width:206pt" width="275">Thermo Fisher Scientific</td>
			<td class="xl70" style="width:89pt" width="119">EN0531</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:275pt;height:15.75pt" width="366">N-Ethylmaleimide</td>
			<td class="xl65" style="width:206pt" width="275">Sigma</td>
			<td class="xl70" style="width:89pt" width="119">E3876&#160;</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:275pt;height:15.75pt" width="366">Sodium Dodecyl Sulfate (SDS)</td>
			<td class="xl65" style="width:206pt" width="275">AMRESCO</td>
			<td class="xl70" style="width:89pt" width="119">M107</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:275pt;height:15.75pt" width="366">Nonidet P-40 Substitute (NP-40)</td>
			<td class="xl65" style="width:206pt" width="275">AMRESCO</td>
			<td class="xl70" style="width:89pt" width="119">M158</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:275pt;height:15.75pt" width="366">protease inhibitor</td>
			<td class="xl65" style="width:206pt" width="275">Roche</td>
			<td class="xl71" style="width:89pt" width="119">5892970001</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:275pt;height:15.75pt" width="366">Mouse Immunoglobulin G (IgG)</td>
			<td class="xl65" style="width:206pt" width="275">Santa Cruz Biotechnology</td>
			<td class="xl71" style="width:89pt" width="119">sc-2025</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl68" height="21" style="height:15.75pt">Rb antibody</td>
			<td class="xl65" style="width:206pt" width="275">Cell Signaling Technology</td>
			<td class="xl71" style="width:89pt" width="119">#9309</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl68" height="21" style="height:15.75pt">Protein A/G-Sepharose Beads</td>
			<td class="xl65" style="width:206pt" width="275">Santa Cruz Biotechnology</td>
			<td class="xl71" style="width:89pt" width="119">sc-2003</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl68" height="21" style="height:15.75pt">Lipofectamine-2000&#160;</td>
			<td class="xl65" style="width:206pt" width="275">Thermo Fisher Scientific</td>
			<td class="xl71" style="width:89pt" width="119">11668019</td>
			<td class="xl68"></td>
		</tr>
		<tr height="23" style="height:17.25pt">
			<td class="xl65" height="23" style="width:275pt;height:17.25pt" width="366">Nickel Nitrilotriacetic Acid (Ni-NTA) Agarose Beads</td>
			<td class="xl65" style="width:206pt" width="275">Qiagen</td>
			<td class="xl71" style="width:89pt" width="119">30230</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:275pt;height:15.75pt" width="366">Imidazole</td>
			<td class="xl65" style="width:206pt" width="275">Sigma</td>
			<td class="xl71" style="width:89pt" width="119">I0250</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl68" height="21" style="height:15.75pt">4%-20% Gradient SDS-PAGE Gel</td>
			<td class="xl65" style="width:206pt" width="275">BIO-RAD</td>
			<td class="xl71" style="width:89pt" width="119">4561096</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl68" height="21" style="height:15.75pt">Polyvinylidene Difluoride (PVDF) Membrane</td>
			<td class="xl65" style="width:206pt" width="275">Millipore</td>
			<td class="xl71" style="width:89pt" width="119">IPVH00010</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:275pt;height:15.75pt" width="366">Tween-20</td>
			<td class="xl65" style="width:206pt" width="275">AMRESCO</td>
			<td class="xl71" style="width:89pt" width="119">M147</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:275pt;height:15.75pt" width="366">Tubulin antibody</td>
			<td class="xl65" style="width:206pt" width="275">Abmart</td>
			<td class="xl71" style="width:89pt" width="119">M30109</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:275pt;height:15.75pt" width="366">SUMO1 antibody</td>
			<td class="xl65" style="width:206pt" width="275">Thermo Fisher Scientific</td>
			<td class="xl71" style="width:89pt" width="119">33-2044</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:275pt;height:15.75pt" width="366">GFP antibody</td>
			<td class="xl65" style="width:206pt" width="275">Abmart</td>
			<td class="xl71" style="width:89pt" width="119">M20004</td>
			<td class="xl68"></td>
		</tr>
		<tr height="23" style="height:17.25pt">
			<td class="xl65" height="23" style="width:275pt;height:17.25pt" width="366">Horseradish Peroxidase (HRP) secondary antibody</td>
			<td class="xl65" style="width:206pt" width="275">Jackson ImmunoResearch Laboratories</td>
			<td class="xl71" style="width:89pt" width="119">715-035-150</td>
			<td class="xl68"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:275pt;height:15.75pt" width="366">enhanced chemiluminescence (ECL) Kit</td>
			<td class="xl65" style="width:206pt" width="275">Thermo Fisher Scientific</td>
			<td class="xl71" style="width:89pt" width="119">32106</td>
			<td class="xl68"></td>
		</tr>
	</tbody>
</table>

in vivo detection and analysis of rb protein sumoylation in human cells

The post-translational modifications of proteins are critical for the proper regulation of intracellular signal transduction. Among these modifications, small ubiquitin-related modifier (SUMO) is a ubiquitin-like protein that is covalently attached to the lysine residues of a variety of target proteins to regulate cellular processes, such as gene transcription, DNA repair, protein interaction and degradation, subcellular transport, and signal transduction. The most common approach to detecting protein SUMOylation is based on the expression and purification of recombinant tagged proteins in bacteria, allowing for an in vitro biochemical reaction which is simple and suitable for addressing mechanistic questions. However, due to the complexity of the process of SUMOylation in vivo, it is more challenging to detect and analyze protein SUMOylation in cells, especially when under endogenous conditions. A recent study by the authors of this paper revealed that endogenous retinoblastoma (Rb) protein, a tumor suppressor that is vital to the negative regulation of the cell cycle progression, is specifically SUMOylated at the early G1 phase. This paper describes a protocol for the detection and analysis of Rb SUMOylation under both endogenous and exogenous conditions in human cells. This protocol is appropriate for the phenotypical and functional investigation of the SUMO-modification of Rb, as well as many other SUMO-targeted proteins, in human cells.

To detect endogenous Rb SUMOylation during cell cycle progression, this study first synchronized HEK293 cells at five different stages of the cell cycle (G0, early G1, G1, S, and G2/M) as described in the protocol section of this paper. The quality of synchronization was confirmed by using the nucleic acid stain with propidium iodide followed by flow cytometry analysis (Figure 1). Next, the cells were collected and lysed by denaturing RIPA buffer.The SUMO pro...

Watch this Scientific Journal Video about In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells at JoVE.com

In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells

Small ubiquitin-related modifier (SUMO) family proteins are conjugated to the lysine residues of target proteins to regulate various cellular processes. This paper describes a protocol for the detection of retinoblastoma (Rb) protein SUMOylation under endogenous and exogenous conditions in human cells.

In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells

The post-translational modifications of proteins are critical for the proper regulation of intracellular signal transduction. Among these modifications, ...

The overall goal of this experiment is to detect retinoblastoma proteins SUMOylation under endogenous and exogenous conditions in human cells. Small ubiquitin-related modifier, or SUMO, family proteins are conjugated to the lysine residues of target proteins to regulate various cellular processes. Usually, it's more challenging to start a protein SUMOylation in cells due to the complexity of SUMOylation in vivo.This experiment detects the SUMOylation of both endogenous and exogenous Rb proteins. Though this method can provide insight into the SUMOylation of Rb proteins, it's also appropriate for the detection of many other SUMO target protein in cells. To begin, add 25 milliliters of DMEM containing one percent penicillin streptomycin, and incubate the HEK293 cells at 37 degrees Celsius for 72 hours.Wash the cells off the plate using three milliliters of ice cold PBS. Then transfer the cells into a new five milliliter tube. To obtain G1-phase cells at the end of the G0 synchronization process, remove the DMEM and add back fresh growth medium, thus allowing the HEK293 cells to re-enter the cell cycle.Then collect the cells at different G1 phases for cell cycle analysis and further SUMO assay. To synchronize the HEK293 cells at the S-phase, use the double thymidine block method. First, grow the HEK293 cells to a confluency of 50%Then wash the cells with pre-warmed PBS.Add growth medium supplemented with 2.5 millimolar thymidine for 18 hours as the first block. Remove the thymidine-containing medium and wash the cells twice with pre-warmed PBS. Add fresh growth medium for 14 hours to release the cells from the block.After releasing the cells from the first block, discard the medium with a pipette. Then add growth medium supplemented with 2.5 millimolar thymidine for another 18 hours as the second block before conducting an analysis of the cell cycle and Rb SUMOylation. For the G2, or M phase synchronization, plate approximately 15 million HEK293 cells on a 15-centimeter dish with 25 milliliters of growth medium, and grow for 24 hours.Then add nicotisol to the medium until a final concentration of 400 nanograms per milliliter is obtained. Finally, incubate the cells for 16 hours before conducting the analysis of the cell cycle by flow cytometry as described in the text protocol. Lyse the synchronized HEK293 cells by gently re-suspending them in one milliliter of ice cold radioimmunoprecipitation assay, or RIPA, lysis buffer containing freshly added isopeptidase inhibitor to block SUMO proteases and stabilize SUMO conjugates.Further homogenize the cells on ice by passing them through a 21 gauge needle attached to a two milliliter syringe 10 times. Then incubate the cells on ice for five minutes. Next, centrifuge the cell lysates at 18000 times G for 30 minutes at four degrees Celsius.Following centrifugation, transfer the supernatants to new 1.5 milliliter microcentrifuge tubes. To the supernatants, add one microgram of non-specific mouse immunoglobulin G of the same species and isotype as the monoclonal Rb antibody. Also, add 20 microliters of 50 percent protein AG sephyroslurry, then incubate the mixture for one hour at four degrees celsius with gentle rotation.Centrifuge the samples at three thousand times g for three minutes at four degrees celsius. Carefully collect the supernatants without disturbing the beads and transfer them to new one point five milliliter micro centrifuge tubes. To each of the samples, add five microliters of rb primary antibody and 40 microliters of 50 percent protein ag sephyroslurry then incubate the samples overnight at four degrees celsius with gentle rotation.After centrifuging, wash the beads with one milliliter of the rifa buffer. Mix the tubes well by rotation at four degrees celsius for 15 minutes. Then collect the beads by low speed centrifugation as before and discard the supernatants.After performing the wash four times, resuspend the beads in 30 microliters of one x sodium dodecalsulfate polyachrylymide gel electrophoresis loading buffer and mix well. Incubate the tubes at 100 degrees celsius in a heat block for 10 minutes. Centrifuge the samples at 12 thousand times g for one minute to pellet the beads.Finally, carefully collect the supernatants without disturbing the beads and transfer them to one point five milliliter microcentrifuge tubes. Perform cell culture transfection and cell lysis of HEK293 cells as described in the text protocol. Then wash 25 microliters of 50 percent nickel nicrolotriacetic acid agoros beads with ripa buffer in a one point five milliliter micro centrifuge tube for each sample.Collect the beads by centrifugation at three thousand times g for three minutes at four degrees celsius. Add one molar emytozol to each sample to obtain a final concentration of 10 millimolar. Then add each sample to the tube containing the prepared agoros beads.Incubate the beads and the lysates for two hours at four degrees celsius with gentle rotation. Then spin the beads at three thousand times g for three minutes at four degrees celsius. Following centrifugation, carefully remove the supernatants by pipeting.To avoid bead loss, do not aspirate the beads dry. Next, wash the beads with one milliliter of wash buffer containing 20 millimolar emytozol. Mix the tubes well by rotation at four degrees celsius for 15 minutes before collecting the beads by spinning.After the final wash, add 30 microliters of the Eleusian buffer containing 250 millimolar emitozol to each sample and flick to mix. Then incubate the samples for 20 minutes at four degrees celsius to elute the proteins. Mix each sample with six x SDS page loading buffer.Then incubate the tubes at 100 degrees celsius in a heat block for 10 minutes. Following centrifugation, load the immunoprecipitation or pull-down samples onto four percent to 20 percent gradient SDS page gels. Conduct electrophoresis at 120 volts for 90 minutes to separate the proteins.After the cell cycle synchronization and immunoprecipitation of the endogenous rb species, the presence of the SUMO signal was specifically detected by an anti SUMO one antibody at the early G1 phase. Representative results show the forced SUMOylation of the rb protein by directly fusing the SUMO E2 enzyme UBC9 to it's C terminal. Note the slowest migrating SUMOylated rb protein.By using the SUMO deficient mutant of rb, K720R, the presence of rb SUMOylation in cells is further confirmed. So in this procedure, many other SUMO target proteins can be analyzed in cultured cells. In most cases only a very small portion of a substrate is SUMOylated, thus while attempting this procedure it's important to optimize the efficiency of immunoprecipitation.

in-vivo-detection-analysis-rb-protein-sumoylation-human

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7.2K ビュー.  Eye and ENT Hospital of Fudan University. The overall goal of this experiment is to detect retinoblastoma proteins SUMOylation under endogenous and exogenous conditions in human cells. Small ubiquitin-related modifier, or SUMO, family proteins are conjugated to the lysine residues of target proteins to regulate various cellular processes. Usually, it's more challenging to start a protein SUMOylation in cells due to the complexity of SUMOylation in vivo.This experiment detects the SUMOylation of both endogenous and exogenous Rb...

ビデオ: In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells

Small ubiquitin-related modifier (SUMO) family proteins are conjugated to the lysine residues of target proteins to regulate various cellular processes. This paper describes a protocol for the detection of retinoblastoma (Rb) protein SUMOylation under endogenous and exogenous conditions in human cells.

in vitro and in vivo detection of mitophagy in human cells, c. elegans, and mice

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and...

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

in-vivo detection of protein-protein interactions on micro-patterned surfaces

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

This video shows experiments with subsequent analysis of protein-protein interactions by the use of micro-patterned surfaces. The approach offers the possibility to detect protein interactions in living cells and combines high throughput capabilities with the possibility to extract quantitative information.

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

protein purification technique that allows detection of sumoylation and ubiquitination of budding yeast kinetochore proteins ndc10 and ndc80

Protein Purification Technique that Allows Detection of Sumoylation and Ubiquitination of Budding Yeast Kinetochore Proteins Ndc10 and Ndc80

This manuscript describes the detection of sumoylation and ubiquitination of kinetochore proteins, Ndc10 and Ndc80, in the budding yeast Saccharomyces...

analysis of scap n-glycosylation and trafficking in human cells

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

We describe a modified method for membrane fraction isolation from human cells and sample preparation for the detection of SCAP N-glycosylation and total protein by using western blot. We further introduce a GFP-labeling method to monitor SCAP trafficking using confocal microscopy. This protocol can be used in regular biology...

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

sumoylation assay: an in vitro technique to detect the sumoylation status of substrate proteins by immunoblotting

This video demonstrates the in vitro method for SUMOylation of substrate proteins using a sequential enzyme cascade. Further, the SUMOylated status of the protein is identified using the electrophoresis and immunoblotting...

SUMOylation Assay: An In Vitro Technique to Detect the SUMOylation Status of Substrate Proteins by Immunoblotting

imaging protein-protein interactions in vivo

Imaging Protein-protein Interactions in vivo

This protocol describes how to image protein-protein interactions using a FRET-based proximity...

Imaging Protein-protein Interactions in vivo

in vitro and in vivo bioluminescence reporter gene imaging of human embryonic stem cells

In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells

With the growing interest in stem cell therapies, molecular imaging techniques are ideal for monitoring stem cell behavior after transplantation. Luciferase reporter genes have enabled non-invasive, repetitive assessment of cell survival, location, and proliferation in vivo. This video will demonstrate how to track hESC proliferation in a living...

the c-fos protein immunohistological detection: a useful tool as a marker of central pathways involved in specific physiological responses in vivo and ex vivo

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Here, we present a protocol based on c-FOS protein immunohistological detection, a classical technique used for the identification of neuronal populations involved in specific physiological responses in vivo and ex...

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

detection of protein ubiquitination

Detection of Protein Ubiquitination

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in...

Next, add 20 microliters of 2x SDS-PAGE with loading buffer to stop the reaction. Then, heat the samples at 95 degrees Celsius for 5 minutes to denature the...

development, expansion, and in vivo monitoring of human nk cells from human embryonic stem cells (hescs) and induced pluripotent stem cells (ipscs)

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

This protocol describes the development, expansion, and in vivo imaging of NK cells derived from hESCs and...

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

rapid detection of neurodevelopmental phenotypes in human neural precursor cells (npcs)

Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells NPCs

Neurodevelopmental processes such as proliferation, migration, and neurite outgrowth are often perturbed in neuropsychiatric diseases. Thus, we present protocols to rapidly and reproducibly assess these neurodevelopmental processes in human iPSC-derived NPCs. These protocols also allow the assessment of the effects of relevant growth factors and therapeutics on NPC...

Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells (NPCs)

detection of dna breaks in dividing human cells by neutral comet assay

Author Spotlight: Unveiling the Role of SNF2L in Replication Fork Stability and Genome Duplication

This protocol is designed to investigate the extent of DNA damage occurring during DNA replication. Under neutral conditions, the induction of DNA breaks can be readily assessed within a short time frame. Additionally, the protocol is adaptable to other cell types and various replication stress...

Detection of DNA Breaks in Dividing Human Cells by Neutral Comet Assay

optimized analysis of in vivo and in vitro hepatic steatosis

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Here, optimized methods to generate in vivo and in vitro models of hepatic steatosis and to analyze the steatotic phenotypes and related physiological parameters are described.

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

in vitro synthesis of modified mrna for induction of protein expression in human cells

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

In this video article, we describe the in vitro synthesis of modified mRNA for induction of protein expression in...

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

intracerebral transplantation and in vivo bioluminescence tracking of human neural progenitor cells in the mouse brain

Intracerebral Transplantation and In Vivo Bioluminescence Tracking of Human Neural Progenitor Cells in the Mouse Brain

We describe the intraparenchymal transplantation of human neural progenitor cells transduced with a dual reporter vector expressing luciferase-green fluorescent protein (GFP) in the mouse brain. After transplantation, the luciferase signal is repeatedly measured using in vivo bioluminescence and GFP-expressing grafted cells identified in brain sections using fluorescence microscopy.

Intracerebral Transplantation and In Vivo Bioluminescence Tracking of Human Neural Progenitor Cells in the Mouse Brain

digital analysis of immunostaining of zw10 interacting protein in human lung tissues

Digital Analysis of Immunostaining of ZW10 Interacting Protein in Human Lung Tissues

ZW10 interacting protein (ZWINT) participates in the mitotic spindle checkpoint and the pathogenesis of carcinoma. Here, we introduce a methodology of the immunostaining of ZWINT in human lung cancer tissues, followed by the digital scanning of whole slides and image analysis. This methodology can provide high-quality digital images and reliable...

in vivo vascular permeability detection in mouse submandibular gland

In Vivo Vascular Permeability Detection in Mouse Submandibular Gland

In the present protocol, the endothelial barrier function of the submandibular gland (SMG) was evaluated by injecting different molecular weighted fluorescent tracers into the angular veins of test animal models in vivo under a two-photon laser-scanning...

In Vivo Vascular Permeability Detection in Mouse Submandibular Gland

detection of the ph-dependent activity of escherichia coli chaperone hdeb in vitro and in vivo

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

This study describes biophysical, biochemical and molecular techniques to characterize the chaperone activity of Escherichia coli HdeB under acidic pH conditions. These methods have been successfully applied for other acid-protective chaperones such as HdeA and can be modified to work for other chaperones and stress...

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative medicine. However, direct injection of hESCs, and cells differentiated from hESCs, into living organisms has thus far been hampered by significant cell death, teratoma formation, and host immune rejection. Understanding the in vivo hESC behavior after transplantation requires novel imaging techniques to longitudinally monitor hESC localization, proliferation, and viability. Molecular imaging has given investigators a high-throughput, inexpensive, and sensitive means for tracking in vivo cell proliferation over days, weeks, and even months. This advancement has significantly increased the understanding of the spatio-temporal kinetics of hESC engraftment, proliferation, and teratoma-formation in living subjects.

A major advance in molecular imaging has been the extension of noninvasive reporter gene assays from molecular and cellular biology into in vivo multi-modality imaging platforms. These reporter genes, under control of engineered promoters and enhancers that take advantage of the host cell s transcriptional machinery, are introduced into cells using a variety of vector and non-vector methods. Once in the cell, reporter genes can be transcribed either constitutively or only under specific biological or cellular conditions, depending on the type of promoter used. Transcription and translation of reporter genes into bioactive proteins is then detected with sensitive, noninvasive instrumentation (e.g., CCD cameras) using signal-generating probes such as D-luciferin.

To avoid the need for excitatory light to track stem cells in vivo as is required for fluorescence imaging, bioluminescence reporter gene imaging systems require only an exogenously administered probe to induce light emission. Firefly luciferase, derived from the firefly Photinus pyralis, encodes an enzyme that catalyzes D-luciferin to the optically active metabolite, oxyluciferin. Optical activity can then be monitored with an external CCD camera. Stably transduced cells that carry the reporter construct within their chromosomal DNA will pass the reporter construct DNA to daughter cells, allowing for longitudinal monitoring of hESC survival and proliferation in vivo. Furthermore, because expression of the reporter gene product is required for signal generation, only viable parent and daughter cells will create bioluminescence signal; apoptotic or dead cells will not. 

In this video, the specific materials and methods needed for tracking stem cell proliferation and teratoma formation with bioluminescence imaging will be described.

With the growing interest in stem cell therapies, molecular imaging techniques are ideal for monitoring stem cell behavior after transplantation. Luciferase reporter genes have enabled non-invasive, repetitive assessment of cell survival, location, and proliferation in vivo. This video will demonstrate how to track hESC proliferation in a living mouse.

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of methodological options for addressing molecular interactions in cells have been developed, but most of these methods suffer from being rather indirect and therefore hardly quantitative. On the contrary, a few high-end quantitative approaches were introduced, which however are difficult to extend to high throughput. To combine high throughput capabilities with the possibility to extract quantitative information, we recently developed a new concept for identifying protein-protein interactions (Schwarzenbacher et al., 2008). Here, we describe a detailed protocol for the design and the construction of this system which allows for analyzing interactions between a fluorophore-labeled protein ("prey") and a membrane protein ("bait") in-vivo. Cells are plated on micropatterned surfaces functionalized with antibodies against the bait exoplasmic domain. Bait-prey interactions are assayed via the redistribution of the fluorescent prey. The method is characterized by high sensitivity down to the level of single molecules, the capability to detect weak interactions, and high throughput capability, making it applicable as screening tool.

This video shows experiments with subsequent analysis of protein-protein interactions by the use of micro-patterned surfaces. The approach offers the possibility to detect protein interactions in living cells and combines high throughput capabilities with the possibility to extract quantitative information.

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of ...

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, preventing their identification and analysis through traditional biochemical methods such as co-immunoprecipitation. In this regard, the genetically encodable fluorescent proteins (GFP, RFP, etc.) and their associated overlapping fluorescence spectrum have revolutionized our ability to monitor weak interactions in vivo using F&ouml;rster resonance energy transfer (FRET)1-3. Here, we detail our use of a FRET-based proximity assay for monitoring receptor-receptor interactions on the endothelial cell surface.

This protocol describes how to image protein-protein interactions using a FRET-based proximity assay.

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

Post-translational Modifications (PTMs), such as phosphorylation, methylation, acetylation, ubiquitination, and sumoylation, regulate the cellular function of many proteins. PTMs of kinetochore proteins that associate with centromeric DNA mediate faithful chromosome segregation to maintain genome stability. Biochemical approaches such as mass spectrometry and western blot analysis are most commonly used for identification of PTMs. Here, a protein purification method is described that allows the detection of both sumoylation and ubiquitination of the kinetochore proteins, Ndc10 and Ndc80, in Saccharomyces cerevisiae. A strain that expresses polyhistidine-Flag-tagged Smt3 (HF-Smt3) and Myc-tagged Ndc10 or Ndc80 was constructed and used for our studies. For detection of sumoylation, we devised a protocol to affinity purify His-tagged sumoylated proteins by using nickel beads and used western blot analysis with anti-Myc antibody to detect sumoylated Ndc10 and Ndc80. For detection of ubiquitination, we devised a protocol for immunoprecipitation of Myc-tagged proteins and used western blot analysis with anti-Ub antibody to show that Ndc10 and Ndc80 are ubiquitinated. Our results show that epitope tagged-protein of interest in the His-Flag tagged Smt3 strain facilitates the detection of multiple PTMs. Future studies should allow exploitation of this technique to identify and characterize protein interactions that are dependent on a specific PTM.

This manuscript describes the detection of sumoylation and ubiquitination of kinetochore proteins, Ndc10 and Ndc80, in the budding yeast Saccharomyces cerevisiae.

Post-translational Modifications (PTMs), such as phosphorylation, methylation, acetylation, ubiquitination, and sumoylation, regulate the cellular ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...