Figure 4

Lyn siRNA inhibits EGFR activation, downstream phosphorylation of c-Met but not phosphorylation of ErbB3. (A) Lyn specific siRNA inhibits Lyn and EGFR phosphorylation. Calu3 cells were cultured with 12.5 nM Lyn siRNA and NC siRNA for 72, 96, and 144 hours before lysates were prepared and 30 μg protein from each treatment loaded into SDS-PAGE wells for separation and Western blotting. Blots were probed with anti-phospho-Lyn, anti-Lyn, anti-phospho-EGFR (Y-1068), anti-phospho-ErbB3 (Y-1289), and actin. (B) EGF triggered reexpression and phosphorylation of Lyn was not evident after 144 hours of culture with 12.5 nM Lyn siRNA while NC siRNA had little effect on Lyn or EGFR phosphorylation levels. EGFR phosphorylations were substantially decreased by Lyn siRNA. Serum-starved Calu3 cells were treated with Lyn and NC siRNA, then cultured for 144 hours. EGF at 100 ng/ml was added for the final 10 minutes before lysates were prepared and 30 μg protein from each treatment loaded into SDS-PAGE wells for separation and Western blotting. Multiple blots were probed with antibodies against phospho-Lyn, non-phosphorylated Lyn control, phospho-EGFR (Y-845), phospho-EGFR (Y-1068), non-phosphorylated ErbB control, phospho-Src (Y-416) or β-actin. (C) Lyn siRNA @ 12.5 nM significantly decreased cell survival after 72 hours of culture. Lyn siRNA decreased viability to highly significant levels, p = <0.001 and 0.0433. Some nonspecific decrease in viability with NC siRNA was also evident but did not reach a similar level of significance. Calcein AM uptake was used to measure cell viability during the last 2 hours of culture. Mann Whitney unit analysis test was applied to relative fluorescent units (RFU) data from 10 replicate wells.