Figure 6

p62 depletion under hypoxia is essential for cell survival. (A–C) Hep3B cells were transfected with pcDNA-p62 (p62 O.E.), pcDNA-Nrf2 (Nrf2 O.E.), sh-RNA Siah2 (Siah2 K.D.), siRNA-Parkin (Parkin K.D.), or siRNA-Hsc70 (Hsc70 K.D.). Cells were then incubated under normoxia or hypoxia (O₂ = 1%) for 12 h. p62 protein levels (A), the viability of cells (B), and the number of apoptotic cells (C) were then assessed by immunoblotting, the MTT colorimetric assay, and colorimetric TUNEL assay, respectively. (D and E) Control and p62-knocked down Hep3B cells were transfected with pcDNA-Nrf2, sh-RNA Siah2, siRNA-Parkin, or siRNA-Hsc70 and then incubated under hypoxia (O₂ = 1%) for 12 h. p62 protein levels (D) and the viability of cells (E) were assessed by immunoblotting and the MTT colorimetric assay, respectively. (F) A working model describing the mechanisms responsible for the depletion of p62 under hypoxia involving transcriptional and post-transcriptional regulation by Nrf2 and Parkin-Hsc70, respectively.