Figure 4.

Hypoxia increases EGFR expression and inhibiting EGFR expression prevents hypoxia‐induced arginase II expression and proliferation. (A) EGFR mRNA was greater in hypoxic HeLa cells (n = 3) than in normoxic cells (n = 3). ** hypoxia different from normoxia, P < 0.0001. (B) Representative western blot showing greater EGFR protein levels in hypoxic HeLa cells than in normoxic HeLa cells. (C) Phosphorylated EGFR at tyrosine 845 (pY845EGFR) was greater in hypoxia than in normoxia. Representative western showing hypoxic phosphorylation of tyrosine 845 in EGFR (pY845EGFR). Densitometry data for pY845EGFR normalized to β‐actin and expressed as fold of normoxia (n = 3 for each exposure). * hypoxia different from normoxia, P < 0.01. (D) EGFR siRNA prevented hypoxic EGFR expression. Representative western blot showing that scramble siRNA (SC) had little effect on hypoxic EGFR expression, while the EGFR siRNA (SiR) prevented EGFR expression. (E) The hypoxia‐induced increase in arginase II protein levels was prevented by treatment with an EGFR siRNA. HeLa cells (n = 3 in each group) were either untreated, treated with scramble siRNA, or treated with siRNA against EGFR, after 24 h cells were washed and allowed to recover for 24 h. The cells were placed in either normoxia or hypoxia for 48 h, and protein harvested for western blotting for arginase II. * hypoxia different from normoxia, P < 0.005. # EGFR siRNA different from scramble siRNA, P < 0.002. (F) The hypoxia‐induced increase in viable cell numbers was prevented by the EGFR siRNA. Cells (n = 3 for each group) were either untreated, treated with scramble siRNA, or treated with siRNA against EGFR, after 24 h cells were washed and allowed to recover for 24 h. The cells were then trypanized and equal numbers of cells plated in each well of a 6 well plate. After 48 h viable cells were counted using trypan blue exclusion. * hypoxia different from normoxia, P < 0.005. # EGFR siRNA different from scramble siRNA, P < 0.005.