Fig. 2: Impact of combined loss of ZMAT3, PUMA and p21 on thymocyte transcriptional landscape and survival upon treatment with various apoptotic stimuli.

Thymocytes from mice of the indicated genotypes were isolated and either (A) left untreated (DMSO control), (B) treated with 1 µg/mL ionomycin, (C) exposed to serum deprivation in culture or (D) treated with 1 µg/mL etoposide. Cell viability was assessed after 24 h by Annexin-V/PI staining and FACS analysis. Data are presented as mean ± SEM of Annexin-V-PI- population (live cells) (A) and live cells relative to DMSO treated control samples (B–D). Representative FACS plots are shown to the right of each graph. E Thymocytes were treated with 5 µg/mL etoposide for 6 h, fixed, followed by intracellular staining for activated (i.e. cleaved) caspase-3 (CC3) and then subjected to FACS analysis. Data are presented as ratio of CC3+ cells in treated vs untreated (DMSO control) thymocytes with representative histograms shown below. FMO = fluorescence minus one staining control. Statistical significance was calculated by one-way ANOVA *p = 0.035, ***p = 0.0009, ****p < 0.0001. F, G RNAseq analysis of isolated thymocytes from 8–12 week old wt (N = 4) and Puma^−/−p21^−/−Zmat3^−/− TKO (N = 3) mice. F Heat map showing all significant differentially expressed (DE) genes between thymocytes from wt and TKO mice. Each column represents data from an individual mouse. Data are colour coded by gene expression Z-scores. Arrows indicate genes that regulate TRP53 and are reported to compromise its tumour suppression function (red), known TRP53 target genes (blue) and splicing regulators (green). G Gene-set enrichment analysis (GSEA) on DE genes between thymocytes from wt and TKO mice shows significant overlap with the Mouse Gene Set: MARTINEZ_TP53_TARGETS_UP [65].