We thank the European Union (FEDER funds POCI/01/0145/FEDER/007728 through Programa Operacional Factores de Competitividade - COMPETE) and National Funds (FCT/MEC, Funda&#231;&#227;o para a Ci&#234;ncia e Tecnologia and Minist&#233;rio da Educa&#231;&#227;o e Ci&#234;ncia) under the Partnership Agreement PT2020 UID/QUI/50006/2019&#160;and the projects (3599-PPCDT) PTDC/DTP-FTO/1981/2014 - POCI-01-0145-FEDER-016581. FCT fellowships: SFRH/BD/96189/2013 (S. Gomes). This work was supported by the Compagnia S. Paolo, Turin, Italy (Project 2017.0526) and Ministry of Health, (Project 5x1000, 2015 and 2016; current research 2016). We deeply thank Dr. Teresa L&#243;pez-Arias Montenegro (University of Trento, experimental sciences teaching laboratories) for assistance with video recording.

1. Construction of ADE2 or LUC1 reporter yeast strains containing a specific RE (yAFM-RE or yLFM-RE)
<ol>
	<li>Streak a yAFM-ICORE or yLFM-ICORE strain12,14 (ICORE = I, ISce-I endonuclease under GAL1 promoter; CO = counter selectable URA3; RE = reporter KanMX4 conferring kanamycin resistance; Table 1) from a 15% glycerol stock stored at -80 &#176;C on a YPDA agar plate (Table 2). Let it grow for 2-3 days at 30 &#176;C.</li>
	<li>Take one yeast colony from the fresh plate (no more than 3 weeks old) and place it in a small flask containing 5 mL of YPDA (Table 2). Incubate at 30 &#176;C overnight, shaking at 150-200 rpm.</li>
	<li>The next day, to remove all traces of dextrose, pellet the cells for 2 min at 3,000 x g and discard the supernatant by inversion of the tube. 
	NOTE: Perform all centrifugations in this protocol at room temperature (RT).</li>
	<li>Resuspend the cell pellet in 30-50 mL of pre-warmed CM (complete media) containing galactose (Table 2) and incubate for 4 h at 30 &#176;C, shaking at 150-200 rpm (necessary for the induction of I-SceI).</li>
	<li>Centrifuge the cells for 2 min at 3,000 x g and discard the supernatant by inversion of the tube.</li>
	<li>Resuspend the cell pellet in 30-50 mL of sterile water. Repeat step 1.5.</li>
	<li>Resuspend the cell pellet in 10 mL of sterile water. Repeat step 1.5.</li>
	<li>Resuspend the cell pellet in 5 mL of sterile LiAcTE (Table 3), an ionic solution that favors DNA uptake. Repeat step 1.5.</li>
	<li>Resuspend the cell pellet in 250 &#181;L of sterile LiAcTE and transfer the cells to a 1.5 mL tube. Repeat step 1.5 and resuspend the cell pellet in 300-500 &#181;L of sterile LiAcTE.</li>
	<li>During the washes, denature a 10 mg/mL solution of salmon sperm DNA carrier for 10 min at 100 &#176;C and chill immediately on ice to maintain it as single-stranded DNA.</li>
	<li>In a separate 1.5 mL tube, add 500 picomoles of the desired oligonucleotide (Table 3), 5 &#181;L of the boiled salmon sperm carrier DNA, 300 &#181;L of sterile LiAcTE PEG (Table 3), and 50 &#181;L of the yeast cell suspension (from step 1.9).</li>
	<li>Vortex the tubes for 10 s to mix and incubate for 30 min at 30 &#176;C, shaking at 150-200 rpm. Place the 1.5 mL tubes on the side to favor shaking.</li>
	<li>Heat shock the yeast cells for 15 min at 42 &#176;C in a heating block, then centrifuge the cells for 20 s at 10,000 x g. Remove the supernatant and resuspend the cells in 1 mL of sterile water.</li>
	<li>Spread 100 &#181;L of the cell suspension on YPDA agar plate and incubate (upside down) for 1 day at 30 &#176;C. To ensure well-separated colonies are obtained, also spread 100 &#181;L of a 1:10 dilution.</li>
	<li>The next day, replica plate using sterile velvets onto CM agar plate containing dextrose and 5-FOA (Table 2). Consider a second replica plate on a new plate if many cells are transferred (i.e., some growth of URA3 cells).</li>
	<li>Three days later, replica plate on non-selective YPDA and YPDA containing G418 (an aminoglycoside antibiotic similar to kanamycin) agar plates (Table 2), marking each plate to facilitate their subsequent comparison. Incubate the plates overnight at 30 &#176;C.</li>
	<li>The next day, identify the candidate reporter strains from colonies that are G418-sensitive but grow on YPDA plates (e.g., yLFM- or yAFM-RE colonies). Streak the identified colonies (3-6 colonies) on a new YPDA plate to obtain single colony isolates and let them grow for 2 days at 30 &#176;C.</li>
	<li>Patch single yeast colonies on a YPDA plate to isolate the colonies for further analyses. After 24 h at 30 &#176;C, test them by replica plating on a YPGA agar plate (Table 2) that prevent the growth of petite mutants (i.e., respiratory-deficient mutants). At the same time, replica plate on a new YPDA agar plate.</li>
	<li>Test the correct patches (i.e., growth on YPDA and YPGA agar plates; 1-3 colonies) from step 1.18 for the presence of correct oligonucleotide integration by colony PCR. Assemble a reaction mix by adding 5 &#956;L of 10x PCR buffer (1.5 mM MgCl2), 2 &#956;L of 10 picomoles/&#956;L primers (Table 3), 4 &#956;L of 2.5 mM dNTPs, 0.25 &#181;L of 5 U/&#956;L Taq polymerase, and water to a final volume of 50 &#956;L. Multiply the reaction mix for the number of yeast colonies that need to be screened and aliquot 50 &#956;L into each PCR tube. Using a pipette, add a very small amount of yeast cells from the YPDA agar plate into a single PCR reaction mix.</li>
	<li>Perform the PCR reaction with the following program: 94 &#176;C for 8 min followed by 35 cycles of denaturation for 1 min at 94 &#176;C, primers annealing for 1 min at 55 &#176;C, and extension for 2 min at 72 &#176;C.</li>
	<li>After the reaction is completed, load an aliquot of the PCR reaction (about one-tenth the volume) on an agarose gel to check the correct size (~500 bp) .</li>
	<li>Sequence the PCR product after purification with a commercial kit to confirm the integration of the desired RE sequence using the same primers of step 1.19.</li>
	<li>After the validation of the correct sequence, make a 15% glycerol stock of yAFM-RE or yLFM-RE strain culture (in YPDA) and store it at -80 &#176;C.</li>
</ol>
2. Evaluation of P53 protein transactivation ability using the qualitative color-based ADE2 yeast assay
<ol>
	<li>Repeat steps 1.1 and 1.2 using the yAFM-RE strain (Table 1).</li>
	<li>The day after, dilute the cell culture (1:10) in 30-50 mL of pre-warmed YPDA and continue to incubate at 30 &#176;C by shaking until the OD600nm reaches 0.8-1.0 (~2 h).</li>
	<li>Repeat steps 1.5-1.10.</li>
	<li>In a separate 1.5 mL tube, add 300-500 ng of yeast P53 (or control) expression vector (Table 4), 5 &#181;L of the boiled salmon sperm DNA carrier, 300 &#181;L of sterile LiAcTE PEG, and 50 &#181;L of yeast cell suspension.</li>
	<li>Repeat steps 1.12 and 1.13 but resuspend the cell pellet in 300 &#181;L of sterile water.</li>
	<li>Spread 100 &#181;L of the cell suspension on synthetic selective (for P53 expression or control vector) plates containing dextrose as a carbon source and high amount of adenine (Table 2), then incubate (upside down) at 30 &#176;C for 2-3 days.</li>
	<li>Streak single yeast transformant colonies (2-6 streaks per plate) on a new selective plate and let them grow overnight at 30 &#176;C.</li>
	<li>The day after, using sterile velvets replica plate onto new selective plates that allow for the assessment of the color phenotype (i.e., plates containing dextrose as carbon source but limiting the amount of adenine ; Table 2). Incubate the plates upside down at 30 &#176;C for 3 days. Optionally, to evaluate temperature sensitivity of P53 protein, incubate at three different temperatures for 3 days: 24 &#176;C, 30 &#176;C, and 37 &#176;C. 
	NOTE: The same streak can be replica plated multiple times.</li>
	<li>To evaluate the P53 protein transactivation ability, check the color-based phenotype of yeast colonies and compare the P53 protein phenotype with respect to P53 wild-type and empty vector phenotypes.</li>
</ol>
3. Evaluation of P53 protein transactivation ability using the quantitative luminescence based LUC1 yeast assay
<ol>
	<li>Transform yeast cells with P53 (or control) expression vectors (Table 4) using the LiAc based method described in protocol 2. Use yLFM-RE strain (Table 1).</li>
	<li>Patch single transformants on a new selective plate with the high amount of adenine containing glucose as the carbon source and let them grow at 30 &#176;C overnight. For each transformation type, make 5-7 different patches.</li>
	<li>After overnight growth, resuspend a small amount of yeast cells using a sterile toothpick or pipette tip from the plate in synthetic selective medium containing dextrose or raffinose as the carbon source (200 &#181;L final volume in a transparent 96 well plate, with a round or flat bottom). If the experiment requires inducible P53 expression, add galactose to the raffinose medium to modulate the level of induction (Table 2). 
	NOTE: These cell suspensions should have an OD600nm of about 0.4 and no higher than 1.</li>
	<li>Measure the absorbance of each well at OD600nm after inducible P53 expression (4-8 h at 30&#176;C with 150-200 rpm shaking) using a multilabel plate reader. Make sure the cell suspensions are homogeneous by mixing each well with a multichannel pipette.</li>
	<li>Transfer 10-20 &#181;L of cell suspension from the transparent 96 well plate into a white 384 (or 96) well plate and mix with an equal volume (10-20 &#181;L) of lysis buffer. Incubate for 10-15 min at RT on a shaker (150-200 rpm) to achieve permeabilization of the cell to the luciferase substrate.</li>
	<li>Add 10-20 &#181;L of Firefly luciferase substrate and measure the light units (LU) by a multi-label plate reader.</li>
	<li>To determine P53 protein transactivation ability, normalize the LUs of each well to the corresponding OD600nm (relative light unit, RLU). Calculate average RLU and standard deviation from 3-4 patches of yeast transformant colonies.</li>
	<li>Compare the P53 protein transactivation data with respect to P53 wild-type and empty vector, either by subtracting the values obtained with the empty vector or by dividing by the values obtained with the empty vector (i.e., computing fold of induction). 
	NOTE: The P53 transactivation activity can be also evaluated using the same experimental set-up in the presence of P53-interacting proteins (i.e., MDM2 and MDMX) and/or including drug treatment.</li>
</ol>
4. Evaluation of P53 protein growth inhibition using the yeast phenotypic assay
<ol>
	<li>Transform yeast cells with P53/MDM2/MDMX (or control) expression vectors (Table 4) using the LiAc-based method described in section 2. Use the CG379 strain (Table 1) and spread yeast transformants on minimal selective plates (Table 2).</li>
	<li>Grow transformed cells in minimal selective medium (Table 2) to approximately 1 OD600nm.</li>
	<li>Dilute yeast cells to 0.05 OD600nm in the selective induction medium (Table 2), and optionally, add a chosen small molecule to the appropriate concentration (or solvent only) to test its efficacy in reactivating mutant P53 or in inhibiting MDM2-/MDMX-P53 interactions.</li>
	<li>Incubate cells at 30 &#176;C under continuous orbital shaking (200 rpm) for approximately 42 h (time required by negative control yeast to reach mid-log phase, about 0.45 OD600nm).</li>
	<li>Spot 100 &#181;L aliquots of yeast cell cultures on minimal selective plates (Table 2).</li>
	<li>Incubate for 2 days at 30 &#176;C.</li>
	<li>Measure yeast growth by counting the number of colonies obtained in the 100 &#181;L culture drops (colony forming unit, CFU counts). For example, calculate the mutant reactivating effect of compounds considering the growth of wild-type P53 expressing yeast as the maximal possible effect (set to 100%), while the growth of cells expressing mutant P53 (but exposed to solvent control) represents the zero level of reactivation.</li>
</ol>

The authors declare no conflict of interest.

Yeast-based assays have proven useful to investigate various aspects of P53 protein functions. These assays are particularly sensitive for evaluating P53 transactivation potential towards variants of RE target sites, including the evaluation of functional polymorphisms. The use of color reporters as well as miniaturization of the luciferase assay result in cost-effective and relatively scalable assays. Also, the growth inhibition test is potentially amenable to being used in chemical library screening, automating the qua...

Transcription is an extremely complex process involving dynamic, spatial, and temporal organization of transcription factors (TFs) and cofactors for the recruitment and modulation of RNA polymerases on chromatin regions in response to specific stimuli1. Most TFs, including the human P53 tumor suppressor, recognize specific cis-acting elements in the form of DNA sequences called response elements (REs), which consist of single (or multiple) unique motifs ~6-10 nucleotides long. Within these motifs, individual positions may show various degrees of variability2, usually summarized by position weight matrices (PWM) or logos3,4.
The yeast S. cerevisiae is a suitable model system for studying different aspects of human proteins through complementation assays, ectopic expression, and functional assays, even when an orthologous yeast gene is not present5,6,7. Due to the evolutionary conservation of basal components of the transcriptional system8, many human TFs (when ectopically expressed in yeast cells) can modulate the expression of a reporter gene by acting through promoters engineered to contain appropriate REs. The transcription model system presented here for human P53 is characterized by three major variables whose effects can be modulated: 1) the modality of expression and type of P53, 2) the RE sequence controlling P53-dependent transcription, and 3) the type of reporter gene (Figure 1A).
Concerning the modality of P53 expression, S. cerevisiae allows the choice of inducible, repressible, or constitutive promoters9,10,11. In particular, the inducible GAL1 promoter allows basal (using raffinose as a carbon source) or variable (by changing the amount of galactose in the media) expression of a TF in yeast. In fact, the finely tunable expression represents a critical development for studying not only P53 itself but also other P53 family proteins12,13.
Regarding the type of REs controlling the P53-dependent expression, S. cerevisiae allows the construction of different reporter strains possessing unique differences in the RE of interest in an otherwise isogenic background. This goal is reached using an adaptation of a particularly versatile genome editing approach developed in S. cerevisiae, called delitto perfetto12,14,15,16.
Furthermore, different reporter genes (i.e., URA3, HIS3, and ADE2) can be used to qualitatively and quantitatively evaluate transcriptional activities of human TFs in S. cerevisiae, each with specific features that can be tailored to experimental needs17,18,19,20,21. The expression of these reporter genes confers uracil, histidine, and adenine prototrophy, respectively. The URA3 reporter does not allow the growth of cells in the presence of 5-FOA as well, and thus it can be counterselected. The ADE2 reporter system has the advantage that, besides nutritional selection, it allows the identification of yeast cells that express wild-type (i.e., functional on ADE2 expression) or mutant (i.e.,not functional on ADE2) P53 from the colony color.
For example, yeast cells expressing the ADE2 gene generate normally sized white colonies on plates containing limiting amounts of adenine (2.5-5.0 mg/L), while those that poorly or do not transcribe it appear on the same plate as smaller red (or pink) colonies. This is due to accumulation of an intermediate in the adenine biosynthetic pathway (i.e., P-ribosylamino-imidazole, which has been previously called amino-imidazole ribotide or AIR), which is converted to form a red pigment. The qualitative color based ADE2 reporter gene has since been replaced with the quantitative Firefly Photinus pyralis (LUC1)12,22. More recently, the ADE2 reporter has been combined with the lacZ reporter in an easy-to-score, semi-quantitative, double reporter assay that can be exploited to sub-classify P53 mutants according to their residual level of functionality23.
Fluorescent reporters such as EGFP (enhanced green fluorescent protein) or DsRed (Discosoma sp. red fluorescent protein) have also been used for the quantitative evaluation of transactivation activity associated with all possible missense mutations in the TP53 coding sequence24. Lastly, the chance of combining tunable promoters for P53 allele expression with isogenic yeast strains differing for the RE and/or reporter gene has led to the development of a data matrix that generates a refined classification of cancer-associated and germline mutant P53 alleles25,26,27.
The approaches described above are used to measure the transcriptional activity of the P53 protein. However, the expression of wild-type P53 in the yeast S. cerevisiae28 and Schizosaccharomyces pombe29 can cause growth retardation, which has been associated with cell cycle arrest28,30 or cell death31. In both cases, yeast growth inhibition is triggered by high P53 expression and has been correlated with potential transcriptional modulation of endogenous yeast genes involved in cell growth. Supporting this hypothesis, the loss-of-function mutant P53 R273H did not interfere with yeast cell growth when expressed at similar levels as wild-type P5332. Conversely, the expression in yeast of the toxic mutant P53 V122A (known for higher transcriptional activity compared to wild-type P53) caused a stronger growth inhibitory effect than wild-type P5332.
Additionally, it was demonstrated that human MDM2 was able to inhibit the human P53 transcriptional activity in yeast, promoting its ubiquitination and subsequent degradation33. Accordingly, the ability of human MDM2 and MDMX to inhibit P53-induced yeast growth inhibition was demonstrated32,34. In an additional study, a correlation between P53 transcriptional activity and actin expression levels was established, with the identification of a putative P53 RE upstream on the ACT1 gene in yeast32. Consistently, actin expression was enhanced by wild-type P53 and even more so by P53 V122A, but not by mutant P53 R273H. Conversely, actin expression by P53 decreased in the co-presence of P53 inhibitors MDM2, MDMX, or pifithrin-&#945;&#160;(a small-molecule inhibitor of P53 transcriptional activity), consistent with results based on the yeast-growth assay. Importantly, these results established a correlation between P53-induced growth inhibition and degree of its activity in yeast, which has been also exploited to identify and study small molecules modulating P53 functions28,34,35.

<table border="0" cellpadding="0" cellspacing="0" style="width:790px;" width="789">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:486px;">L-Aspartic acid</td>
			<td style="width:119px;">SIGMA</td>
			<td style="width:123px;">11189</td>
			<td style="width:63px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QIAquick PCR Purification Kit</td>
			<td style="width:119px;">QIAGEN</td>
			<td style="width:123px;">28104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Phenylalanine</td>
			<td style="width:119px;">SIGMA</td>
			<td style="width:123px;">78019</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:486px;">Peptone</td>
			<td style="width:119px;">BD Bacto</td>
			<td>211677</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:486px;">Yeast ex+A2:C26tract</td>
			<td style="width:119px;">BD Bacto</td>
			<td>212750</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Difco Yeast Nitrogen Base w/o Amino Acids and Ammonium Sulfate</td>
			<td style="width:119px;">BDTM</td>
			<td style="width:123px;">233520</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lithium Acetate Dihydrate</td>
			<td style="width:119px;">SIGMA</td>
			<td style="width:123px;">517992</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:486px;">Bacteriological Agar Type A</td>
			<td style="width:119px;">Biokar Diagnostics</td>
			<td style="width:123px;">A1010 HA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:486px;">G418 disulfate salt</td>
			<td style="width:119px;">SIGMA</td>
			<td style="width:123px;">A1720</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ammonium Sulfate</td>
			<td style="width:119px;">SIGMA</td>
			<td style="width:123px;">A2939</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Arginine Monohydro-chloride</td>
			<td style="width:119px;">SIGMA</td>
			<td style="width:123px;">A5131</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:486px;">Adenine Hemisulfate Salt</td>
			<td style="width:119px;">SIGMA</td>
			<td style="width:123px;">A9126</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:486px;">Passive Lysis Buffer 5x</td>
			<td style="width:119px;">PROMEGA</td>
			<td style="width:123px;">E1941</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:486px;">Bright-Glo Luciferase Assay System&#160;</td>
			<td style="width:119px;">PROMEGA</td>
			<td style="width:123px;">E2620</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:486px;">5-FOA</td>
			<td style="width:119px;">Zymo Research</td>
			<td style="width:123px;">F9001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:486px;">D-(+)-Galactose</td>
			<td style="width:119px;">SIGMA</td>
			<td style="width:123px;">G0750</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Glutamic acid</td>
			<td style="width:119px;">SIGMA</td>
			<td style="width:123px;">G1251</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:486px;">Dextrose&#160;</td>
			<td style="width:119px;">SIGMA</td>
			<td style="width:123px;">G7021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Histidine</td>
			<td style="width:119px;">SIGMA</td>
			<td style="width:123px;">H8125</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Isoleucine</td>
			<td style="width:119px;">SIGMA</td>
			<td style="width:123px;">I2752</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Lysine</td>
			<td style="width:119px;">SIGMA</td>
			<td style="width:123px;">L1262</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:486px;">L-Leucine</td>
			<td style="width:119px;">SIGMA</td>
			<td style="width:123px;">L8000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Methionine</td>
			<td style="width:119px;">SIGMA</td>
			<td style="width:123px;">M2893</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PEG</td>
			<td style="width:119px;">SIGMA</td>
			<td style="width:123px;">P3640</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:486px;">D-(+)-Raffinose Pentahydrate</td>
			<td style="width:119px;">SIGMA</td>
			<td style="width:123px;">R0250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Serine</td>
			<td style="width:119px;">SIGMA</td>
			<td style="width:123px;">S4500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Tryptophan</td>
			<td style="width:119px;">SIGMA</td>
			<td style="width:123px;">T0271</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Threonine</td>
			<td style="width:119px;">SIGMA</td>
			<td style="width:123px;">T8625</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Uracil</td>
			<td style="width:119px;">SIGMA</td>
			<td style="width:123px;">U0750</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Valine</td>
			<td style="width:119px;">SIGMA</td>
			<td style="width:123px;">V0500</td>
			<td></td>
		</tr>
	</tbody>
</table>

yeast as a chassis for developing functional assays to study human p53

The finding that the well-known mammalian P53 protein can act as a transcription factor (TF) in the yeast S. cerevisiae has allowed for the development of different functional assays to study the impacts of 1) binding site [i.e., response element (RE)] sequence variants on P53 transactivation specificity or 2) TP53 mutations, co-expressed cofactors, or small molecules on P53 transactivation activity. Different basic and translational research applications have been developed. Experimentally, these approaches exploit two major advantages of the yeast model. On one hand, the ease of genome editing enables quick construction of qualitative or quantitative reporter systems by exploiting isogenic strains that differ only at the level of a specific P53-RE to investigate sequence-specificity of P53-dependent transactivation. On the other hand, the availability of regulated systems for ectopic P53 expression allows the evaluation of transactivation in a wide range of protein expression. Reviewed in this report are extensively used systems that are based on color reporter genes, luciferase, and the growth of yeast to illustrate their main methodological steps and to critically assess their predictive power. Moreover, the extreme versatility of these approaches can be easily exploited to study different TFs including P63 and P73, which are other members of TP53 gene family.

Construction of ADE2 or LUC1 reporter yeast strains
Thedelitto perfettoapproach12,14,15,16 has been adapted to enable the construction of P53 reporter yeast strains (Figu...

Watch this Scientific Journal Video about Yeast As a Chassis for Developing Functional Assays to Study Human P53 at JoVE.com

Yeast As a Chassis for Developing Functional Assays to Study Human P53

Presented here are four protocols to construct and exploit yeast Saccharomyces cerevisiae reporter strains to study human P53 transactivation potential, impacts of its various cancer-associated mutations, co-expressed interacting proteins, and the effects of specific small molecules.

The finding that the well-known mammalian P53 protein can act as a transcription factor (TF) in the yeast S. cerevisiae has allowed for the development of ...

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Cancer Research

9.4K Views.  Ospedale Policlinico San Martino. Presented here are four protocols to construct and exploit yeast Saccharomyces cerevisiae reporter strains to study human P53 transactivation potential, impacts of its various cancer-associated mutations, co-expressed interacting proteins, and the effects of specific small molecules.

Video: Yeast As a Chassis for Developing Functional Assays to Study Human P53

Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased understanding of the molecular events that give rise to glioblastomas, this cancer still remains highly refractory to conventional treatment. Surgical resection of high grade brain tumors is rarely complete due to the highly infiltrative nature of glioblastoma cells. Therapeutic approaches which attenuate glioblastoma cell invasion therefore is an attractive option. Our laboratory and others have shown that tumor associated macrophages and microglia (resident brain macrophages) strongly stimulate glioblastoma invasion. The protocol described in this paper is used to model glioblastoma-macrophage/microglia interaction using in vitro culture assays. This approach can greatly facilitate the development and/or discovery of drugs that disrupt the communication with the macrophages that enables this malignant behavior. We have established two robust coculture invasion assays where microglia/macrophages stimulate glioma cell invasion by 5 - 10 fold. Glioblastoma cells labelled with a fluorescent marker or constitutively expressing a fluorescent protein are plated without and with macrophages/microglia on matrix-coated polycarbonate chamber inserts or embedded in a three dimensional matrix. Cell invasion is assessed by using fluorescent microscopy to image and count only invasive cells on the underside of the filter. Using these assays, several pharmacological inhibitors (JNJ-28312141, PLX3397, Gefitinib, and Semapimod), have been identified which block macrophage/microglia stimulated glioblastoma invasion.

Understanding the malignant behavior of cancer requires creating accurate models of how tumor cells interact with components of the tumor microenvironment, such as macrophages. Here we describe two methods to study glioblastoma cell interaction with tumor associated macrophages and microglia where the effect on glioblastoma invasion is assessed.

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased ...

How to Study Basement Membrane Stiffness as a Biophysical Trigger in Prostate Cancer and Other Age-related Pathologies or Metabolic Diseases

Here we describe a protocol that can be used to study the biophysical microenvironment related to increased thickness and stiffness of the basement membrane (BM) during age-related pathologies and metabolic disorders (e.g. cancer, diabetes, microvascular disease, retinopathy, nephropathy and neuropathy). The premise of the model is non-enzymatic crosslinking of reconstituted BM (rBM) matrix by treatment with glycolaldehyde (GLA) to promote advanced glycation endproduct (AGE) generation via the Maillard reaction. Examples of laboratory techniques that can be used to confirm AGE generation, non-enzymatic crosslinking and increased stiffness in GLA treated rBM are outlined. These include preparation of native rBM (treated with phosphate-buffered saline, PBS) and stiff rBM (treated with GLA) for determination of: its AGE content by photometric analysis and immunofluorescent microscopy, its non-enzymatic crosslinking by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) as well as confocal microscopy, and its increased stiffness using rheometry. The procedure described here can be used to increase the rigidity (elastic moduli, E) of rBM up to 3.2-fold, consistent with measurements made in healthy versus diseased human prostate tissue. To recreate the biophysical microenvironment associated with the aging and diseased prostate gland three prostate cell types were introduced on to native rBM and stiff rBM: RWPE-1, prostate epithelial cells (PECs) derived from a normal prostate gland; BPH-1, PECs derived from a prostate gland affected by benign prostatic hyperplasia (BPH); and PC3, metastatic cells derived from a secondary bone tumor originating from prostate cancer. Multiple parameters can be measured, including the size, shape and invasive characteristics of the 3D glandular acini formed by RWPE-1 and BPH-1 on native versus stiff rBM, and average cell length, migratory velocity and persistence of cell movement of 3D spheroids formed by PC3 cells under the same conditions. Cell signaling pathways and the subcellular localization of proteins can also be assessed.

Here we explain a protocol for modelling the biophysical microenvironment where crosslinking and increased stiffness of the basement membrane (BM) induced by advanced glycation endproducts (AGEs) has pathological relevance.

Here we describe a protocol that can be used to study the biophysical microenvironment related to increased thickness and stiffness of the basement ...

The Colon-26 Carcinoma Tumor-bearing Mouse as a Model for the Study of Cancer Cachexia

Cancer cachexia is the progressive loss of skeletal muscle mass and adipose tissue, negative nitrogen balance, anorexia, fatigue, inflammation, and activation of lipolysis and proteolysis systems. Cancer patients with cachexia benefit less from anti-neoplastic therapies and show increased mortality1. Several animal models have been established in order to investigate the molecular causes responsible for body and muscle wasting as a result of tumor growth. Here, we describe methodologies pertaining to a well-characterized model of cancer cachexia: mice bearing the C26 carcinoma2-4. Although this model is heavily used in cachexia research, different approaches make reproducibility a potential issue. The growth of the C26 tumor causes a marked and progressive loss of body and skeletal muscle mass, accompanied by reduced muscle cross-sectional area and muscle strength3-5. Adipose tissue is also lost. Wasting is coincident with elevated circulating levels of pro-inflammatory cytokines, particularly Interleukin-6 (IL-6)3, which is directly, although not entirely, responsible for C26 cachexia. It is well-accepted that a primary mechanism by which the C26 tumor induces muscle tissue depletion is the activation of skeletal muscle proteolytic systems. Thus, expression of muscle-specific ubiquitin ligases, such as atrogin-1/MAFbx and MuRF-1, represent an accepted method for the evaluation of the ongoing muscle catabolism2. Here, we present how to execute this model in a reproducible manner and how to excise several tissues and organs (the liver, spleen, and heart), as well as fat and skeletal muscles (the gastrocnemius, tibialis anterior, and quadriceps). We also provide useful protocols that describe how to perform muscle freezing, sectioning, and fiber size quantification.

Mice bearing the Colon-26 (C26) carcinoma represent a classical model of cancer cachexia. Progressive muscle wasting occurs in association with tumor growth, over-expression of muscle-specific ubiquitin ligases, and reductions in muscle cross-sectional area. Fat loss is also observed. Cachexia is studied in a time-dependent manner with increasing severity of wasting.

Cancer cachexia is the progressive loss of skeletal muscle mass and adipose tissue, negative nitrogen balance, anorexia, fatigue, inflammation, and ...

Dried Blood and Serum Spots As A Useful Tool for Sample Storage to Evaluate Cancer Biomarkers

Blood sample quality is crucial to ensure accurate downstream analyses such as real-time PCR or ELISA. Correct storage of biological materials is the starting point to achieve reproducible and reliable results. All samples should be treated in the same way from blood collection to storage. Depending on the analyses to be performed, whole blood and serum samples should be stored at -20 &#176;C or -80 &#176;C until use. Blood/serum samples should also be aliquoted to avoid multiple freeze-thawing. Another important issue is the sample conditions during shipment from one laboratory to another. If dry ice is not available or the shipment takes longer than a few days, alternative approaches are needed. One option is to use filter paper for blood collection. Here, we propose a method for blood and serum sample collection that takes advantage of dried blood spots (DBS) and dried serum spots (DSS). We developed the procedure to extract DNA from DBS for the downstream evaluation of some single nucleotide polymorphisms (SNPs) by real time PCR. We also optimized an ELISA assay starting from proteins eluted from DSS. This method can be used with other ELISA assays or procedures evaluating proteins.

This protocol describes a simple and useful method to store peripheral blood and serum/plasma for downstream analyses such as single nucleotide polymorphism (SNP) evaluation and ELISA assay.

Blood sample quality is crucial to ensure accurate downstream analyses such as real-time PCR or ELISA. Correct storage of biological materials is the ...

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include rapid proliferation and pervasive invasion into the normal brain. Recurrence is thought to result from the presence of radio- and chemo-resistant brain tumor stem cells (BTSCs) that invade away from the initial cancerous mass and, thus, evade surgical resection. Hence, therapies that target BTSCs and their invasive abilities may improve the otherwise poor prognosis of this disease. Our group and others have successfully established and characterized BTSC cultures from GBM patient samples. These BTSC cultures demonstrate fundamental cancer stem cell properties such as clonogenic self-renewal, multi-lineage differentiation, and tumor initiation in immune-deficient mice. In order to improve on the current therapeutic approaches for GBM, a better understanding of the mechanisms of BTSC migration and invasion is necessary. In GBM, the study of migration and invasion is restricted, in part, due to the limitations of existing techniques which do not fully account for the in vitro growth characteristics of BTSCs grown as neurospheres. Here, we describe rapid and quantitative live-cell imaging assays to study both the migration and invasion properties of BTSCs. The first method described is the BTSC migration assay which measures the migration toward a chemoattractant gradient. The second method described is the BTSC invasion assay which images and quantifies a cellular invasion from neurospheres into a matrix. The assays described here are used for the quantification of BTSC migration and invasion over time and under different treatment conditions.

Here, we describe live-cell imaging techniques to quantitatively measure the migration and invasion of glioblastoma brain tumor stem cells over time and under multiple treatment conditions.

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

Prostate Organoid Cultures as Tools to Translate Genotypes and Mutational Profiles to Pharmacological Responses

Presented here is a protocol to study pharmacodynamics, stem cell potential, and cancer differentiation in prostate epithelial organoids. Prostate organoids are androgen responsive, three-dimensional (3D) cultures grown in a defined medium that resembles the prostatic epithelium. Prostate organoids can be established from wild-type and genetically engineered mouse models, benign human tissue, and advanced prostate cancer. Importantly, patient derived organoids closely resemble tumors in genetics and in vivo tumor biology. Moreover, organoids can be genetically manipulated using CRISPR/Cas9 and shRNA systems. These controlled genetics make the organoid culture attractive as a platform for rapidly testing the effects of genotypes and mutational profiles on pharmacological responses. However, experimental protocols must be specifically adapted to the 3D nature of organoid cultures to obtain reproducible results. Described here are detailed protocols for performing seeding assays to determine organoid formation capacity. Subsequently, this report shows how to perform drug treatments and analyze pharmacological response via viability measurements, protein isolation, and RNA isolation. Finally, the protocol describes how to prepare organoids for xenografting and subsequent in vivo growth assays using subcutaneous grafting. These protocols yield highly reproducible data and are widely applicable to 3D culture systems.

Presented here is a protocol to study pharmacological responses in prostate epithelial organoids. Organoids closely resemble in vivo biology and recapitulate patient genetics, making them attractive model systems. Prostate organoids can be established from wildtype prostates, genetically engineered mouse models, benign human tissue, and advanced prostate cancer.

Presented here is a protocol to study pharmacodynamics, stem cell potential, and cancer differentiation in prostate epithelial organoids. Prostate ...

Combined Conditional Knockdown and Adapted Sphere Formation Assay to Study a Stemness-Associated Gene of Patient-derived Gastric Cancer Stem Cells

Cancer stem cells (CSCs) are implicated in tumor initiation, development and recurrence after treatment, and have become the center of attention of many studies in the last decades. Therefore, it is important to develop methods to investigate the role of key genes involved in cancer cell stemness. Gastric cancer (GC) is one of the most common and mortal types of cancers. Gastric cancer stem cells (GCSCs) are thought to be the root of gastric cancer relapse, metastasis and drug resistance. Understanding GCSCs&#160;biology is needed to advance the development of targeted therapies and eventually to reduce mortality among patients. In this protocol, we present an experimental design using a conditional knockdown system and an adapted sphere formation assay to study the effect of clusterin on the stemness of patient-derived GCSCs. The protocol can be easily adapted to study both in vitro and in vivo function of stemness-associated genes in different types of CSCs.

In this protocol, we present an experimental design using a conditional knockdown system and an adapted sphere formation assay to study the effect of clusterin on the stemness of patient-derived GCSCs. The protocol can be easily adapted to study both in vitro and in vivo function of stemness-associated genes in different types of CSCs.

Cancer stem cells (CSCs) are implicated in tumor initiation, development and recurrence after treatment, and have become the center of attention of many ...

Monitoring eIF4F Assembly by Measuring eIF4E-eIF4G Interaction in Live Cells

Formation of the eIF4F complex has been shown to be the key downstream node for the convergence of signalling pathways that often undergo&#160;oncogenic activation in humans. eIF4F is a cap-binding complex involved in the mRNA-ribosome recruitment phase of translation initiation. In many cellular and pre-clinical model of cancers, the deregulation of eIF4F leads to increased translation of specific mRNA subsets that are involved in cancer proliferation and survival. eIF4F is a hetero-trimeric complex built from the cap-binding subunit eIF4E, the helicase eIF4A and the scaffolding subunit eIF4G. Critical for the assembly of active eIF4F complexes is the protein-protein interaction between eIF4E and eIF4G proteins. In this article, we describe a protocol to measure eIF4F assembly that monitors the status of eIF4E-eIF4G interaction in live cells. The eIF4e:4G cell-based protein-protein interaction assay also allows drug induced changes in eIF4F complex integrity to be accurately and reliably assessed. We envision that this method can be applied for verifying the activity of commercially available compounds or for further screening of novel compounds or modalities that efficiently disrupt formation of eIF4F complex.

Here, we present a protocol to measure eIF4E-eIF4G interaction in live cells that would enable the user to evaluate drug induced perturbation of eIF4F complex dynamics in screening formats.

Formation of the eIF4F complex has been shown to be the key downstream node for the convergence of signalling pathways that often undergo&#160;oncogenic ...

Using the E1A Minigene Tool to Study mRNA Splicing Changes

mRNA processing involves multiple simultaneous steps to prepare mRNA for translation, such as 5&#180;capping, poly-A addition and splicing. Besides constitutive splicing, alternative mRNA splicing allows the expression of multifunctional proteins from one gene. As interactome studies are generally the first analysis for new or unknown proteins, the association of the bait protein with splicing factors is an indication that it can participate in mRNA splicing process, but to determine in what context or what genes are regulated is an empirical process. A good starting point to evaluate this function is using the classical minigene tool. Here we present the adenoviral E1A minigene usage for evaluating the alternative splicing changes after different cellular stress stimuli. We evaluated the splicing of E1A minigene in HEK293 stably overexpressing Nek4 protein after different stressing treatments. The protocol includes E1A minigene transfection, cell treatment, RNA extraction and cDNA synthesis, followed by PCR and gel analysis and quantification of the E1A spliced variants. The use of this simple and well-established method combined with specific treatments is a reliable starting point to shed light on cellular processes or what genes can be regulated by mRNA splicing.

This protocol presents a rapid and useful tool for evaluating the role of a protein with uncharacterized function in alternative splicing regulation after chemotherapeutic treatment.

mRNA processing involves multiple simultaneous steps to prepare mRNA for translation, such as 5&#180;capping, poly-A addition and splicing. Besides ...

A Rapid Screening Workflow to Identify Potential Combination Therapy for GBM using Patient-Derived Glioma Stem Cells

The glioma stem cells (GSCs) are a small fraction of cancer cells which play essential roles in tumor initiation, angiogenesis, and drug resistance in glioblastoma (GBM), the most prevalent and devastating primary brain tumor. The presence of GSCs makes the GBM very refractory to most of individual targeted agents, so high-throughput screening methods are required to identify potential effective combination therapeutics. The protocol describes a simple workflow to enable rapid screening for potential combination therapy with synergistic interaction. The general steps of this workflow consist of establishing luciferase-tagged GSCs, preparing matrigel coated plates, combination drug screening, analyzing, and validating the results.

The glioma stem cells (GSCs) are a small fraction of cancer cells which play essential roles in tumor initiation, angiogenesis, and drug resistance in ...