FIGURE 4.

Effect of Trx on p53 expression and DNA binding in hypoxia. A, HCT116 cells (vector (Vec), Trx, or dnTrx) were exposed to normoxia or hypoxia (0. 2% O₂) for 24 or 48 h. Cell lysates were analyzed for p53 expression and p53 (Ser-15) phosphorylation by Western analysis as detailed under “Experimental Procedures.” Top panel, expression of total p53; middle panel, expression of phospho-p53 (Ser-15); bottom panel, expression of β-actin. B, HCT116 cells (vector, dnTrx, or Trx) were exposed to 21% oxygen or 0.2% O₂ + 5% CO₂ + 94% N₂ for 24 h. Cells were harvested; nuclear extract was prepared, and p53 EMSA was performed as described under “Experimental Procedures.” C, MCF-7 cells (vector, Trx, of dnTrx cells) were exposed to normoxia or hypoxia as mentioned for A, and p53 EMSA was performed as detailed under “Experimental Procedures.” D, MCF-7 clones were treated and processed as described in A. Origin recognition complex-2 (ORC-2) in the nuclear lysates was detected as a loading control. E, MCF-7 clones were exposed to 21% oxygen or 8 or 16 h of hypoxia, and p53 expression was analyzed using Western analysis as mentioned in B. F, various clones of HCT116 cells were exposed to normoxia or hypoxia (0.2% 24 h), and p53 Western analysis was performed as described under “Experimental Procedures.” G, various clones of MCF-7 cells were exposed to normoxia or hypoxia (0.2% 24 h), and p53 Western analysis was performed as described under “Experimental Procedures.”