Figure 5
FGFR4 silencing downregulates the anti-apoptotic proteins c-FLIP and Bcl-2 and reduces pSTAT3 activity in colon cancer cells. (a) Western blotting was used to measure expression of FGFR4, PARP, Pro-Caspase 8, Caspase 8 p18, FLIPL, FLIPS, Bcl-2, BCLXL, Bax, MCL1 and XIAP at 24–72 h following transfection with 5 nM siFGFR4. (b) Q-PCR of c-FLIP and Bcl-2 expression following transfection of HCT116 cells with siFGFR4 for 24–48 h. (c) Annexin V/PI flow cytometric analysis was used to measure apoptosis in HCT116 parental cells and the overexpressing cell line, FL17, following 72 h FGFR4 silencing. (d) Western blotting was used to measure expression of FGFR4, pSTAT3, tSTAT3, pAKT, tAKT, pERK and tERK following transfection of 5 nM siFGFR4 for 24–48 h in HCT116, 48 h in RKO and 10 nM siFGFR4 for 48 h in LS174T cells. (e) A luciferase construct STAT3 reporter assay was used to measure STAT3 activity in HCT116 at 24 and 48 h following transfection with 5 nM siFGFR4. (f) Western blotting was used to measure the expression of pSTAT3, tSTAT3, c-FLIPL and Bcl-2. Equal loading was assessed by probing for GAPDH or actin. Significance was assessed by two-way analysis of variance (c) or Student’s t-test (b and d). Results are presented as mean values±S.E.M. for three replicate experiments (*P<0.05, **P<0.01, ***P<0.001)
