Fig 2: Noxa levels influence the sensitivity of GSC-ECL to BH3-mimetics. (a) Cells were transfected with either NT siRNA or Noxa specific siRNA and exposed 48 h to ABT-263 (1 μM) or WEHI-539 (1 μM), combined or not with chemotherapeutic agents. Percentage of cell death (PI+ cells) was determined by flow cytometry in GSC-ECLs. Bar charts show the mean ± S.E.M. of three independent experiments. Student's t-test was conducted to detect significant differences *P < 0.05, **P < 0.01, ***P < 0.001. (b) mRNA levels of Noxa were analyzed by RT-qPCR after 48 h of incubation with the indicated treatments. Bar charts show the mean ± S.E.M. of three independent experiments. Student's t-test was conducted to detect significant differences between untreated and treated cells. *P < 0.05, **P < 0.01, ***P < 0.001. (c) Protein levels of Noxa were analyzed by Western blot after 48 h of incubation with the indicated treatments. Actin was used as loading control. Full-length images are shown in Supplementary Figs. S22 and S23. Bar graphs representing densitometric quantifcation of bands are depicted in Supplementary Fig. S1. (d) G03 and G09 cells were transfected with either NT plus Noxa siRNAs or Bcl-2 plus Bcl-w plus Noxa siRNAs and exposed 48 h to WEHI-539 (1 μM). Percentage of cell death (PI+ cells) was determined by flow cytometry in GSC-ECLs. Bar charts show the mean ± S.E.M. of three independent experiments. For each cell line, the Y-axis scale was set based on the range of the values in the corresponding data set.

Fig 3: Identification of differentially expressed genes in p53‐mRNA‐transfected OVCAR‐8 cells compared to mock‐transfected counterparts. (A) Heatmap of normalized gene expression represented by z scores of OVCAR‐8 cells treated with mock (EGFP mRNA) or p53‐mRNA from three independent RNA‐seq experiments (Student's t tests or Welch tests, depending on the variance, corrected by FDR q adjusted P‐value < 0.05 and absolute FC > 1.5). (B) Volcano plot showing the DEGs in the RNA‐seq between OVCAR‐8 cells treated with EGFP vs. p53‐mRNA (red dots = upregulated genes, blue dots = down‐regulated genes). (C) Selected overrepresented Ingenuity pathways from pathway enrichment analysis of DEGs with FDR‐adjusted P‐values < 0.05 and FC > 1.5. Pathways with a positive z score, representing a global upregulation of the pathway genes, are depicted in orange; pathways with a negative z score, indicating a global gene downregulation in the pathway, are depicted in blue; and pathways with no specific pattern are depicted in gray. (D) Heatmap of all genes corresponding to the “p53 signaling pathway” from IPA, indicating the P‐value, FDR‐adjusted P‐value and FC for the comparison of EGFP with p53‐mRNA. Genes with validation by WB are highlighted in red. (E) Cell lysates were subjected to WB for p53 signaling pathways using p21, MDM2, Puma, p73, Fas, and CDK1 antibodies. CDK1, cyclin‐dependent kinase 1; DEG, differentially‐expressed gene; EGFP, enhanced green fluorescent protein; Fas, tumor necrosis factor receptor superfamily member 6; FC, fold change; FDR, false‐discovery rate; IPA, Ingenuity Pathway Analysis; MDM2, double minute 2 homolog protein; Puma, p53 upregulated modulator of apoptosis; Noxa, NADPH oxidase activator 1; p53, tumor protein p53; RNA‐seq, RNA sequencing.

Fig 4: Liposomal transfection of in vitro‐transcribed WT p53‐mRNA induces cell cycle arrest and apoptosis in the HGSOC cell line OVCAR‐8. (A) OVCAR‐8 cells were transfected with increasing amounts of liposomal IVT Flag‐tagged WT p53‐mRNA. Cell lysates were subjected to WB using Flag‐ and β‐Actin‐antibodies. (B) The dose‐dependent cell cycle distribution of Nocodazole‐synchronized cells is represented as a bar graph (upper panel; n = 3). Cell lysates were subjected to WB using Flag‐, Cyclin E1‐, Cyclin B1‐, PLK1‐, and β‐Actin‐antibodies (lower panel). (C) Cell lysates were subjected to WB for key mediators of cell cycle regulation using Flag‐, p53 (pS20)‐, p53‐, p21‐, p27‐, p16‐, PLK1‐, Cyclin A1‐, Cyclin B1‐, Cyclin E1‐, Aurora A‐, CDK1‐, and β‐Actin‐antibodies (upper panel). The same cell lysates were subjected to WB for regulators of apoptosis using PARP‐, Caspase‐3‐, Puma‐, Noxa‐, and Fas‐antibodies (lower panel). (D) Cells were mock‐ or 0.5 μg p53‐mRNA‐transfected, and proliferation was measured using a CellTiter‐Blue Cell Viability assay, 24 h post‐treatment. ** P < 0.01, *** P < 0.001, Student's t‐test, unpaired and two‐tailed. (E) The dose‐dependent Caspase‐3/7 activities were determined using a Caspase‐Glo 3/7 assay. *** P < 0.001, Student's t‐test, unpaired and two‐tailed. (F) Graphical representation of Annexin V positive (%) cells. FACS analysis of transfected cells, gated for Annexin V positive (early apoptotic), and Annexin V/7AAD positive (late apoptosis) populations is demonstrated. n = 3 for each dose; ** P < 0.01, *** P < 0.001. (G) Representative images show the number of 3D colonies (dose‐dependent) on day 10 (left panel). The dose‐dependent distribution of colonies is represented as bar graph (right panel; n = 3). * P < 0.05, *** P < 0.001, Student's t‐test, unpaired and two‐tailed. (H) Graphical representation of the cell cytotoxity caused by increasing concentrations of transfected p53 mRNA. n = 3 for each concentration; * P < 0.5, **P < 0.01.Aurora‐A, Aurora Kinase A; CDK1, cyclin‐dependent kinase 1; FACS, Fluorescence‐Activated Cell SortingA; Fas, Tumor necrosis factor receptor superfamily member 6; HGSOC, High‐Grade Serous Ovarian Cancer; IVT, in vitro‐transcribed; Noxa, NADPH oxidase activator 1; p53, tumor protein p53; PARP, Poly (ADP‐ribose) polymerase; PLK1, Polo‐like kinase 1; WB, western blot.

Fig 5: Inhibiting BCL-XL sensitises medulloblastoma cells to the apoptosis-inducing effects of cisplatin treatment.A DR_MOMP calculated the η score for ONS76 and UW228 cell lines following in silico administration of the indicated concentrations of ABT-199, S63845 or WEHI-539. B BH3 profiling was carried out to determine the basal anti-apoptotic dependencies of ONS76 (left) and UW228 (right) cell lines using the indicated BH3 peptides. Briefly, BAD targets BCL-2 and BCL-XL, HRK targets BCL-XL, and NOXA and A12 are specific for MCL-1. BIM is a measure of overall priming as it promiscuously interacts with all anti-apoptotic proteins. Data are expressed as the mean ± SEM of N = 3 independent experiments performed in triplicate, normalised to DMSO (0% mitochondrial depolarisation) and FCCP (100% mitochondrial depolarisation). C Cell viability in ONS76 (left) and UW228 (right) was measured following 48 h treatment with increasing concentrations of cisplatin combined with the indicated concentrations of WEHI-539 using the WST-1 viability assay. Data are expressed as mean ± SEM of N = 3 independent experiments performed in duplicate. D ONS76 and UW228 cells were treated with the indicated concentrations of WEHI-539 for 48 h and viability was determined using the Wst-1 viability assay. Data points represent the mean ± SEM of 3 independent experiments carried out in duplicate. The x-axis is not continuous. E Combination index values were calculated based on the above data (C) to assess synergy between cisplatin and WEHI-539 in ONS76 (left) and UW228 (right) cells. A score <1 indicates synergy between the two agents, while <0.8 indicates strong synergy. F Flow cytometry was used to assess the number of AnnexinV+PI- + AnnexinV+PI+ cells following 48 h treatment with cisplatin (20 µM), or cisplatin with BH3 mimetics (1 µM). The effect of treatment with WEHI-539 (1 µM) alone was also investigated. Data are expressed as mean of N = 3 independent experiments, ±SEM. Two-way ANOVA followed by Tukey’s multiple comparisons test was used for statistical analysis, whereby *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ONS76 (G) and UW228 (H) cells were transiently transfected with non-targeting control siRNA (siControl) or targeted siRNA against BCL-XL (siBCL-XL). BCL-XL protein expression was assessed with Western blotting 48 h post-transfection to verify successful knockdown, with GAPDH used as a loading control. Representative blots of N = 3 independent experiments are shown. Cells were subsequently treated with cisplatin (20 µM) for a further 24 h, and Annexin-V/PI staining followed by flow cytometry analysis was used to determine the proportion of apoptotic cells. Data are expressed as mean of N = 3 independent experiments ±SEM. Data were analysed using One-Way ANOVA with Tukey’s multiple comparison test, whereby **p < 0.01, ****p < 0.0001.