Figure 4. siRNA silencing and interruption of septin dynamics with FCF decrease ErbB2 levels in gastric cancer cells.

AGS cells were transiently transfected with septin-2-specific or control (ctr) siRNA or AGS and HGE-20 cells were incubated with FCF for 12 hours. Western blot was performed on clarified lysates. ErbB2 protein levels were decreased with siRNA silencing of septin-2. Use of FCF led to a similar decrease in ErbB2 protein in both cell lines. The structurally similar EGFR was not affected, demonstrating specificity of response. β-actin was used as a loading control (A). Immunofluroescence with ErbB2 antibodies in HGE-20 cells showed accumulation of ErbB2 in intracellular compartments following 6 hour incubation with FCF, with overall decreased expression and minimal intracellular appearance of ErbB2 at 12 hours, suggestive of intracellular degradation (B). Arrows in the images point to intracellular accumulation of ErbB2. Numbers below images indicate the intensity of total fluorescence as % of control as calculated by analyzing at least 10 microscopic fields for each condition using Zen 2009 software. n=3 independent experiments, errors or error bars represent standard deviation (s.d.), *-significant difference from condition without siRNA or FCF, p<0.05, Student’s t-test.