Effect of hypoxia and/or paclitaxel or epirubicin on Mcl-1 mRNA expression level and protein expression. MDA-MB-231 cells were incubated under normoxic (N) or hypoxic (H) conditions with or without paclitaxel (tax, 50 μM) or epirubicin (epi, 10 μM) for 16 hours (A, B). 8 h post transfection with c-jun siRNA (siRNA) or RISC-free control siRNA (RF) (50 nM, 24 h), cells were incubated under normoxic condition with or without paclitaxel (tax, 50 μM) (C). (A) After incubation, total RNA has been extracted and retro-transcribed in cDNA. A real time PCR has been performed with specific primers for Mcl-1 and for RPL13A, a house-keeping gene. Results are expressed in induction level by comparison with the reference condition, normoxia. (B) Mcl-1 was detected by immunofluorescence using specific Mcl-1 antibody. Nuclei were stained with Topro-3 (in blue). Observation was performed in semi-quantitative confocal microscopy. (C) Mcl-1 was detected in total cell extracts by western blotting, using specific antibody. α-tubulin was used to assess the total amount of proteins loaded on the gel.