Figure 4

Analysis of TMEM45A gene expression level and effect of TMEM45A silencing on TMEM45A expression and caspase 3 activity in HepG2 cells. HepG2 cells were incubated under normoxic (N) or hypoxic (H) conditions with or without etoposide (etop, 50 μM) for 16 hours, 8 h post transfection with TMEM45A siRNA (siRNA) or RISC-free control siRNA (RF) (50 nM, 24 h). (A) After transfection and incubation, total RNA has been extracted and retro-transcribed in cDNA. A real time PCR has been performed with specific primers for TMEM45A and for RPL13A, a house-keeping gene. Results are expressed in induction level by comparison with the reference condition, normoxia. (B) After transfection and incubation, caspase 3 activity was assayed by measuring free AFC released from the cleavage of the caspase 3 specific substrate Ac-DEVD-AFC. Results are expressed in fluorescence intensity, as mean ± 1 SD (n = 3). Statistical analyses were determined independently for the 3 subgroups without siRNA, with TMEM45A siRNA (siRNA) and with RISC-free control siRNA (RF) ; N.S. = non significantly different from control (N, N siRNA or N RF), ** = significantly different from control (p < 0.01), *** = significantly different from control (p < 0.001) ; N.S. ₍₁₎ = no significant difference between N etop and H etop, ### = significant difference between N etop and H etop (p < 0.001), N.S. = no significant difference between ₍₂₎ N etop and N etop RF, ₍₃₎ N etop and N etop siRNA, or ₍₄₎ H etop and H etop RF, ··· = significant difference between H etop and H etop siRNA (p < 0.001).