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. 2012 Aug 3;7(8):e42291. doi: 10.1371/journal.pone.0042291

Figure 2. ASM knockdown attenuates the RGDfV-induced changes in ceramides and sphingomyelins.

Figure 2

ECV-304 cells were transfected with siRNA as described in Methods. A) Messenger RNA extracted from HBMEC 48 hrs after transfection with ASM1, ASM2, ERK, or control siRNA was analyzed for ASM expression by qRT-PCR (n = 6; p<0.001 for ASM1, p<0.001 for ASM2 and p = 0.54 for ERK by unpaired t-test for each siRNA compared to non-silencing control). B) ASM activity in extracts of ECV-304 72 hrs after ASM-siRNA transfection compared to NC controls (Echelon Biosciences kit). Shown are mean±SEM; n = 3; p<0.001 for ASM1 and for ASM2, by unpaired t-test. C–D) Change in ceramide (panel C) and sphingomyelin (panel D) in extracts of ECV-304 cells after NC, ASM siRNA #1 or ASM siRNA #2, that were then incubated with vehicle or RGDfV (5 µg/ml; 24 hrs) and analyzed by mass spectrometry. Bars represent mean±SEM fold change from control in one of two experiments with similar results, each of which was performed in total of 3 biological triplicates. Absolute lipid values are provided in Table 1.