Figure 4

Functional evaluation of IMP3 in hCPC in homeostasis and in response to oxidative damage. (a) Comparative RT-qPCR expression analysis of IGF2R, IMP2 and IMP3 in hCPC1 and MSC19. (b), (c) Confirmation of downregulation of IMP3 in hCPC (1,3) transfected with sIMP3 compared with a negative control (siNeg) and untransfected control cells (control), by RT-qPCR relative to the expression of GusB (b) and western blot (c); bottom panel corresponds to the quantification, relative to tubulin, on the representative western blot (upper panel); 'full-length blots/gels' are presented in Supplementary Figure S5 on line. All samples were analyzed 48 h post-transfection. (d) Evaluation of cell viability in hCPC (1,3) transfected with sIMP3 compared with a negative control (siNeg) and untransfected control cells (control), by DAPI staining, evaluated 48 h post-transfection. (e) Analysis of the effects of IMP3 downregulation on hCPC (1,3) response to oxidative damage induced by H₂O₂. hCPC control, siIMP3- or siNeg-transfected cells were exposed to H₂O₂ (500 μM) for 48 h; then cultures were stained with AnnexinV/propidium iodide (Anex.V/P.I.) and homeostatic viable (Anex.V − /PI −), apoptotic (Anex.V + /PI −), late apoptotic (Anex.V + / PI +) or necrotic (Anex.V −/PI +) cells were quantified by cytometry. Assays were performed three times and data expressed as mean ± SD; black lines summarize p-values (*** < 0.002, ** < 0.02, * < 0.05; one-way ANOVA analysis of variance followed by the Bonferroni correction for multiple comparison).