Fig 3. CerS6 is a target of MTX in mediation of antiproliferative effect.

(A) Silencing CerS6 by siRNA rescues A549 cells from cytotoxic effect of MTX while silencing CerS4 does not change the drug toxicity. Cells were transfected with scrambled siRNA, CerS6 siRNA or CerS4 siRNA (25 pmol), MTX was added 6 h later and live cells were assessed by MTT assay 48 h after the beginning of MTX treatment. Data represent an average of two independent experiments, each performed in quadruplicates with error bars representing SD. Student’s t test was performed for statistical analysis (difference in cell number between control and CerS6-silenced cells were statistically significant with p<0.005; there were no significant differences in cell number between control and CerS4-silenced cells). Panels on the right show the efficiency of CerS6 and CerS4 silencing (evaluated at 48 h of MTX treatment). (B) Levels of ceramide in control A549 cells (Cntr, transfected with scrambled siRNA), MTX-treated cells transfected with scrambled siRNA (MTX) and MTX-treated cells after CerS6 silencing (CerS6 siRNA/MTX, transfected with CerS6 siRNA). Differences between the groups were analyzed by one-way ANOVA and asterisks indicate statistically significant changes (p<0.05). (C) Silencing of CerS6 by siRNA protects cancer cells from MTX cytotoxic effect. Student’s t-test was performed and the differences between MTX effect in control and CerS6-silenced cells were statistically significant (p<0.01). (D) MTX (10 nM) leads to elevation of CerS6 but not of CerS4 in A549 cells.