Figure 5. Inhibition of c-Abl abrogates the RGDfV-induced increase in ASM mRNA and ASM activity.

A–B) ECV-304 cells were treated with STI-571 (c-Abl inhibitor; 10 µM) or vehicle starting 2 hrs before adding RGDfV (5 µg/ml) or vehicle for additional 24 hrs. (A) ASM mRNA (real time qRT-PCR; mean±SEM of 4 independent experiments performed in 2 replicates), (B) ASM activity (Echelon Biosciences kit; mean±SEM of 2 independent experiments performed in 4 replicates). C–D) ASM mRNA (real time qRT-PCR relative to GAPDH) of ECV-304 transfected with non-specific non-silencing negative control siRNA (NC) or c-Abl siRNA (48 hrs) and incubated with RGDfV (5 µg/ml) or vehicle for 24 hrs. Shown are mean±SEM, n = 4. Representative efficacy of c-Abl siRNA knockdown (western blot) is shown in Panel D. E) Change in ceramide content in extracts from cells incubated with STI-571 or siRNA-c-Abl and their controls with/without RGDfV as in panels A–D was analyzed by mass spectrometry. Bars represent mean±SEM fold change in ceramides C₁₆ and C₁₈ from four (STI-571) and three (siRNA, either non-silencing controls NC, or c-Abl) independent experiments. Changes in other ceramides were not significant. P-values were calculated using paired t-tests. F–G) Phospho-c-Abl (Y412) and total c-Abl (western blotting) in lysates of ECV-304 transfected with NC, ASM1 or ASM2 siRNA as in Fig. 3 and treated 24 hrs with RGDfV (5 µg/ml) or vehicle. GAPDH served as loading control. Panel G shows a typical experiment and panel F shows mean±SEM of phospho-c-Abl (Y412) relative to total c-Abl from densitometry of three experiments. ASM mRNA level in these experiments was 28±1.2% of the non-silencing control samples for siRNA-ASM1 and 11.4±0.9% for siRNA-ASM2.