Figure 4
Effect of siRNA-mediated down regulation of HIF-1α in hPSCs cell viability and death upon 1% O2 hypoxia induction. H9 hESCs and FN2.1 hiPSCs were transfected with negative control non-targeting siRNA (NT siRNA) (20 nM) or HIF-1α siRNA (20 nM) and then: (a) mRNA expression levels of HIF-1α were analyzed by RT-qPCR at 24 and 48 h post siRNAs transfection. RPL7 expression was used as normalizer. Graph shows mean + SEM mRNA fold induction relative to NT siRNA transfectants arbitrarily set as 1 from three independent experiments. Statistical analysis was done by Student’s t-test, (***) p < 0.001 versus NT siRNA. (b) Expression levels of HIF-1α were analyzed by western blot in H9 and FN2.1 cells at 48 h post siRNAs transfection. HIF-1α was stabilized by hypoxia (1% O2 for 24 h starting at 24 h post siRNAs transfection) treatment. ACTIN was used as loading control. Representative blots are shown (full-length images are presented in Supplementary Fig. S12). (c) Representative histograms of Propidium iodide (PI) stained H9 and FN2.1 unfixed cells at 48 h post siRNA transfection. 24 h 1% O2 hypoxia treatment was started at 24 h post siRNA transfection. The percentage of PI-positive cells (late apoptotic or necrotic) was determined by flow cytometric analysis. Mean + SEM from three independent experiments are graphed. Statistical analysis was done by Student’s t-test, (*) p < 0.05 vs. normoxia. (d) Histograms show the percentage of surviving cells assessed by Trypan blue exclusion method at 48 h post siRNA transfection. 24 h after transfection cells were incubated for 24 h at 1% O2. Mean + SEM from three independent experiments is shown. Statistical analysis was done by Student’s t-test, (*) p < 0.05, (**) p < 0.01, and (***) p < 0.001 vs. NT siRNA.
