Fig. 8: Ceramide synthases, especially CerS2, drive oncogenic K-Ras-induced senescence and mediate the increase of senescence induced by SK1 downregulation. | Cell Death & Disease

Fig. 8: Ceramide synthases, especially CerS2, drive oncogenic K-Ras-induced senescence and mediate the increase of senescence induced by SK1 downregulation.

From: Targeting sphingosine kinase 1 (SK1) enhances oncogene-induced senescence through ceramide synthase 2 (CerS2)-mediated generation of very-long-chain ceramides

Fig. 8

A Western blot analysis of p21 expression in cell lysates from cells overexpressing GV K-Ras. Cells were plated and treated the next day with Fumonisin B1 (FB1) or Myriocin (Myr) for 72 h at 1 μM or 100 nM final, respectively. Cells were then lysed and the protein level of p21 was determined. B Western blot analysis of p21 expression in cell lysates from cells overexpressing GV K-Ras. Cells were plated and transfected the next day with control (AS) or CerS1-6 siRNA (20 nM) for 72 h. Cells were then lysed and the protein level of p21 was determined. C Western blot analysis of p21 expression in cell lysates from cells overexpressing GV K-Ras. Cells were plated and treated the next day with Fumonisin B1 (FB1) and transfected with control (AS) or SK1 siRNA (20 nM) for 72 h. Cells were then lysed and the protein level of p21 was determined. D Western blot analysis of p21 expression in cell lysates from cells overexpressing GV K-Ras. Cells were plated and cotransfected the next day with control (AS) or SK1 siRNA or control (AS) and CerS2 siRNA or SK1 and CerS2 siRNA (20 nM) for 72 h. Cells were then lysed and the protein level of p21 was determined. E, F SL analysis using tandem LC/MS/MS in cells overexpressing mutant GV K-Ras. Cells were plated and cotransfected the next day with control (AS) or SK1 siRNA or control (AS) and CerS2 siRNA or SK1 and CerS2 siRNA (20 nM) for 72 h and then scraped in a lipid extraction buffer. SL levels were quantified and normalized to the amount of total lipid phosphate (N = 3, data are presented as mean ± SEM, two-way ANOVA with Dunnett’s multiple comparisons tests, § means p < 0.05 comparing the group GV SK1 siRNA to the group GV SK1 + CerS2 siRNA). G Cell growth analysis of cells overexpressing mutant GV K-Ras. Cells were plated and treated the next day with FB1 (1 μM). A number of viable cells were evaluated after trypsinization every other day until 5 days in culture (N = 5, data are presented as mean ± SEM, two-way ANOVA with Tukey’s multiple comparisons test, * means p < 0,05 comparing the group tested to Vector control).

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