Effect of hypoxic, acidic and serum-depleted culture conditions on CD26 expression in HCT-116 cells. A) Effect of hypoxia (1% O2) on CD26 protein expression in cells at sub-confluence. Cells at ~50% confluence were incubated under hypoxic or normoxic conditions. Cells were harvested at different time points and protein expression was analyzed by Western blotting as described in Materials and Methods. B) Effect of hypoxia on CD26 protein expression at 3 days post-confluence. Cells were grown to 3 days post-confluence, then incubated under hypoxic or normoxic conditions for 24 hours. C) Expression of HIF-1α at sub- and postconfluence. Cells were grown for 8 days, from sub-confluence through 5 days post-confluence and protein expression was analyzed by Western blotting. D) Enhanced CD26 expression in parental HCT-116 cells compared to HCT-116HIF1-α-/- cells. Whole cell lysates (24 μg) were run on gels and analyzed as described previously. Cells were held at confluence for 3 days (lane 1), 1 day (lane 2), or were sub-confluent (lane 3). E) The effect of acidic conditions on CD26 expression. Cells were grown to 3 days post-confluence as described above, and then incubated in regular 10% FBS medium (pH 7.3) or in acidic medium supplemented with 25 mM MES (pH 6.2) for 24 hours. F) The effect of hypoxia, acidic medium, and serum depletion was examined in cells at sub-confluence. G) The effect of switching cells from serum-depleted culture conditions to 10% FBS medium was evaluated. Cells were grown to ~25% confluence (lane #1) and then cultured in regular 10% FBS medium (lane #2) or serum-depleted medium (lane #3) for 24 hours. Cells cultured in serum-free medium for 24 hours were re-cultured in regular 10% FBS medium for another 24 hours (lane# 4). Cell confluence at this point was still ~90%. All data discussed above are representative of at least three independent experiments.