Figure 1.

Primary mouse cell lines respond to genotoxic stress through p53-dependent, caspase-2 mediated proteolysis of SK1 to alter the balance of pro- and anti-growth sphingolipids. (a) Mouse embryonic fibroblasts (MEFs) expressing WT p53 or with p53 knocked out, were treated with indicated doses of UV radiation (J/m²), cultured for 48 hours, and then whole cell lysate was examined by (a) Western blotting with antibodies to the indicated proteins, or, (b) harvested in RLT buffer, prepared for qRT-PCR, with enzyme expression normalized to β-actin expression for each reaction performed in triplicate, or, (c) WT MEFs were pre-treated with 20 uM z-VDVAD-fmk (caspase-2 inhibitor) dissolved in DMSO or DMSO control in fresh media and incubated for one hour prior to treatment with 0 or 20 J/m² UV radiation, incubated for 48 hours before being harvested by scraping on ice using Tris-Triton lysis buffer supplemented with protease and phosphatase inhibitors from Sigma for western blotting with antibodies to the indicated proteins (n=3), or (d) harvested for sphingolipidomic analysis to measure S1P and C₁₆-ceramide by LC/MS, expressed as pmol lipid per mg protein ± SEM (n=3, and *p < 0.05 by Student’s t test relative to WT 0 UV).