Figure 4.

Representative fluorescent intensity flow cytometric histograms show the effect of LAM or FN with or without EF on membrane expression of EGFR on CECs. Isotype-matched negative control, OX21 as primary mAb, background fluorescence was similar with secondary Ab as negative control. M1 (positively stained cells) was set where the expression of gated events was <1% in isotype-matched negative control. (A) Control (without substratum coating, no EF) cells showed a population of positively stained cells, region M1, which can be further divided into a low peak (region M2) representing a subpopulation of low EGFR expression (EGFR^Low) and a higher peak (region M3) representing a subpopulation of high EGFR expression (EGFR^High) expression. (B) After 12 h in EF (100 mV/mm), positively stained cells increased, as shown by gated events in M1 as total, M2 as EGFR^Low expression cells (p < 0.01), and M3 as EGFR^High expression cells. The cells were cultured on plastic without substratum coating. (C) Withdrawal of serum from culture medium and exposed to EF (100 mV/mm) for 13 h shows a significant decrease in EGFR expression (p < 0.05 for EGFR^High). The cells were cultured on plastic without substratum coating. (D) Addition of EGF (25 ng/ml) in serum-free medium and EF (100 mV/mm) for 13 h; EGFR^Low expression was significantly up-regulated; compare with C. Also see text for detailed numerical data. (E) Significant increases in total EGFR expression and more dramatically in EGFR^High were evident for CECs cultured on substratum of LAM (1 μg/ml) followed by 3 h of EF exposure at 100 mV/mm.