Figure 6.

Effect of c-jun silencing on hypoxia-induced resistance to etoposide-induced apoptosis. HepG2 cells were transfected with 50 nM c-jun siRNA or negative control siRNA for 24 hours. Eight hours later, cells were incubated under normoxic (Nx; 21% O₂) or hypoxic (Hx; 1% O₂) conditions with (Ne-He; 50 µM) or without etoposide (Nx-Hx) for 16 hours. (A and B) At the end of the incubation, total RNA was extracted, submitted to reverse transcription, and then to amplification in the presence of SYBR Green and specific primers for CDKN1A and PUMA. RPL13A was used as the housekeeping gene for data normalization. One experiment of two independent ones is shown here (A, B). (C) At the same time, total extracts were prepared and analyzed by Western blot analysis for p53, PTEN, Bak1, and c-jun using specific antibodies. α-Tubulin was used to assess the total amount of proteins loaded on the gel.