mBax_499 mRNA construct is lethal for hESCs. (a,b) Brightfield images of monolayer hESC cultures and measurement of cell viability and DNA content after (a) 4 h and (b) 24 h of treatment with various mRNAs. Scale bar: 130 µm. Cell viability (red) was evaluated by PrestoBlue assay and normalized to the value of control untreated cells (n = 3). Survival rates (green) were verified through DNA quantification by Hoechst 33342 after the removal of dead cells (n = 3). Values are according to calibration of intensity vs. cell count. (c,d) Observation of DNA fragmentation in selected images of single cells stained with 7AAD taken from ImageStreamX (c) showing brightfield (CH01), fluorescence 480 nm/615 nm (CH05) and SSC (CH06) and its analysis (d) using bright detail intensity R7 feature of positively stained cells for quantification of apoptotic events. (e) Caspase 3 activity of hESCs post treatment with either mBax_499 mRNA, non-lethal mRNA or etoposide (n = 3). (f) Western blot analysis of Bax protein level in hESCs post treatment (with: lanes 1:2 – eGFP mRNA, lanes 3:4 – Bax mRNA, lanes 5:6 – mBax mRNA) and its densitometric analysis (n = 2). Band intensity was normalized to the GAPDH housekeeping protein band intensity. Lane 7 – mBax protein produced by cell-free translation of mBax mRNA. All bar graphs indicate mean (SD). Full-length uncropped blots with low exposure are presented in Supplementary Fig. S1.3. p values were generated using one-way ANOVA with Tukey’s post-hoc test for multiple comparisons. *p < 0.5, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not statistically different.