Figure 1: P53 expression and localization changes during cardiac differentiation
(A) Timeline of human in vitro cardiac differentiation of hiPSCs with indicated small molecules used and the days in which samples were collected for RNA, protein, and immunostaining. Developmental stages are indicated by color underneath their corresponding days. (B) RNA was collected from the indicated time points in A and subjected to qRT-PCR and the results compiled into a heatmap. The genes were grouped temporally based on their expression levels with genes identifying pluripotency (orange), cardiac mesoderm (green), cardiac progenitors (blue), and cardiomyocytes (red). These stages are color coded and correlate back to 2A. (n = 4) (C) qRT-PCR of P53 mRNA expression across the cardiac differentiation. (n = 28). (D) Representative western blot and quantification of P53 protein expression during cardiac differentiation (n = 3). (E) Immunostaining for P53 in human iPS colonies (Tra-1-60+), Day 5-cardiac progenitors (NKX2-5 +), and in Day 15 cardiomyocytes (α-actinin +). Scale bars = 20 μm

Figure 2: Early stages of cardiac differentiation show increased sensitivity to P53 levels. (A) Time line of sample collection during the first 10 days of cardiac differentiation with or without Nutlin-3a exposure in 24 hour increments. (B) Representative western blot of P53 protein levels and cleaved PARP at each day of the differentiation treated either with DMSO (vehicle)or 10 uM of Nutlin-3a for 24 hours. Vinculin is included as a loading control. The black line indicates the samples were run on separate gels (n=2).
(C) After the 24 hour incubation with Nutlin-3a, each day of differentiation was incubated with EdU for 1 hour, stained with a Live- Dead viability dye and then subjected to flow cytometry to assess cell death and proliferation. An increasing concentration of Nutlin- 3a showed a dose-dependent increase in cell death in cells younger than Day 4. Nutlin-3a treatment had little effect on cell viability for cells older than Day 4 (n ≥ 4 each day). (D) The same population of cells in (C) were stained for EdU and DAPI to identify the number of cells in S phase. There was a dose-dependent decrease in the % of EdU+ cells in S phase with increasing Nutlin-3a concentration with the majority of the cells remaining in G1. The amount of proliferating cells also decreased naturally as the cells continued to differentiate (n ≥ 2 each day). (E) Cells at the time points indicated were exposed to a dose curve of Nutlin-3a for 24 hours, after which the drug was removed, and the cells were cultured as normal out to Day 20. They were then stained with a Live- Dead viability dye and cardiac markers cTnI and cTnT to assess cardiomyocyte output. Addition of Nutlin-3a prior to Day 4 caused a dose-dependent decrease in the proportion of cardiomyocytes produced. Addition of Nutlin-3a past Day 5 slightly increased the proportion of cardiomyocytes (n=2) (#: p< 0.05, *: p< 0.01).

Figure 3: RNA-Seq study revealed different gene expression profiles between the control and Nutlin-3a treated samples during hiPSCs cardiac differentiation
(A) Time course RNA-Seq experimental design included 4 time points in both control and Nutlin-3a treated groups: Day 3 Cardiac Mesoderm, Day 5 Cardiac Progenitors, Day 7 Cardiomyocytes (No beating yet), Day 10 Cardiomyocytes (Beating). Each group in each time point had 8 samples except for Day 10 which had 6 samples. Treated groups were treated with 10uM Nutlin-3a for 24 hours. Samples were collected at each time point to extract mRNA for sequencing. (B) PCA was performed on all samples from both groups (orange: Nutlin-3a, green: control). The results revealed a dramatic difference between control and drug- treated groups. (C) The following differential analysis between both groups at all time points highlighted genes displaying variable patterns of differential expression between the two groups (1). K-mean cluster analysis was performed to cluster the genes into different sub-clusters based on their expression patterns, with focus on activated and inhibited gene clusters shown (2). Each gene cluster underwent gene interaction network analysis and function enrichment analysis to investigate their roles in Nutlin-3a treatment (3). At the end, gene expression information, interaction networks, and enriched functions were integrated together to highlight important genes (such as hub genes) (4). The integration results are shown in the following Figures 4 & 5.

Figure 4: Genes activated by Nutlin-3a treatment highlight gene functions involved in cellular oxidant detoxification, apoptosis, DNA damage response related to P53
(A) Gene interaction network lines indicate the gene interactions with wider lines for higher interaction scores from the Gene/Protein Interaction Database (String DB). The bubble size represents the number of interactions connected to that gene. Genes with larger bubbles are hub genes in the interaction network. (B) Top 5 enriched function pathways for Nutlin-3a activated genes. 1: Cellular oxidant detoxification. 2: Regulation of apoptotic process. 3: Response to toxic substance. 4: Negative regulation of protein kinase activity. 5: DNA damage response, signal transduction by P53 class mediator resulting in cell-cycle arrest. (C) Time course gene expression profiles from RNA-Seq data with green for lower expression and red for higher expression. Time course RNA-Seq design included critical time points: Day 3 (D3), 5, 7, and 10. Each time point contained two groups: control (C) and Nutlin-3a-treated group (T). To save space, average expression level of 8 samples in each group was plotted. (D) Gene symbols with genes with more than 10 interaction partners are labeled in red. These red genes are hubs in the interaction network.

Figure 5: Genes inhibited by Nutlin-3a treatment demonstrated functions relating to cell division, mitotic nuclear division and cytokinesis
(A) Gene interaction network lines indicate the gene interactions with wider lines for higher interaction scores from the Gene/Protein Interaction Database (String DB). The bubble size represents the number of interactions connected to that gene. Genes with larger bubbles are hub genes in the interaction network.
(B) The top 5 enriched function pathways inhibited by Nutlin-3a treatment included 1: Cell division, 2: Mitotic nuclear division, 3: Chromosome segregation, 4: Sister chromatid cohesion, 5: Mitotic cytokinesis. (C) Time course gene expression profiles from RNA-Seq data with green for lower expression and red for higher expression. Time course RNA-Seq design included critical time points: Day 3 (D3), 5, 7, and 10. Each time point contained two groups: control (C) and Nutlin-3a-treated group (T). To save space, average expression level of 8 samples in each group was plotted. (D) Gene symbols with genes with more than 30 interaction partners are labeled in red. These red genes are hubs in the interaction network.

Figure 6: Nutlin-3a causes anti-oxidant Sestrin gene increases in cardiomyocytes. RNA-Seq revealed response to oxidative stress as one of the top pathways activated by Nutlin-3a with Sestrin1 and Sestrin2 implicated. These changes were validated by qRT- PCR on the right (n=3) and the original RPKM values on the left ( n ≥ 6)

Supplementary Figure 2: Immunofluorescence images of P53 localization during cardiac differentiation with Nutlin-3a treatment
Cells were treated with DMSO or 10 μM Nutlin-3a for 24 hours then fixed and stained for P53 and an appropriate cell stage marker. (A) Human induced pluripotent stem cell colonies. (B) Day 1 Nanog + cells. (C) Day 5 NKX2-5 + cardiac progenitors. (D) Day 7 cardiomyocytes not yet beating.
(E) Day 11 cardiomyocytes that were spontaneously beating. Treatment with Nutlin-3a in all stages except hiPSC colonies resulting in increased nuclear staining of P53.

Supplementary Figure 3: IIncreased apoptosis in early stages of cardiac differentiation was prevented by addition of a pan-caspase inhibitor
(A) Each time point of the differentiation was incubated with either 10 μM Nutlin-3a for 6, 12, and 24 hours or 10 μM Nutlin-3a with 50 μM ZFAD-FMK, a pan-caspase inhibitor. A representative western blot of cleaved PARP revealed that ZFAD-FMK incubated alongside Nutlin-3a eliminated the presence of cleaved PARP in the early stages of differentiation. (B) Flow cytometry of cells incubated with 10 μM Nutlin-3a for 12 hours or 10 μM Nutlin-3a for 24 hours plus 50 μM ZFAD-FMK. Cells were stained with a Live-Dead viability dye and the number of dead cells quantified. Addition of ZFAD-FMK caused a dramatic reduction in cell death in these early stages of cardiac differentiation.

Supplementary Figure 4: Cardiomyocytes do not undergo apoptosis despite increased P53 and Puma levels. (A) Western blot for pro- and anti-apoptotic protein levels each day of cardiac differentiation after 24 hours of Nutlin-3a treatment. Vinculin was included as a loading control. (n=2) (B) qRT-PCR analysis of pro- and anti-apoptotic genes influenced by P53 signaling. (n=3) (#p<0.05, *p<0.01)