Figure 3
From: S1P lyase regulates DNA damage responses through a novel sphingolipid feedback mechanism
SPL sensitizes cells to apoptosis after DNA damage by depleting S1P and increasing ceramide levels. (a) Total cellular S1P content in control (open bar) and SPLhi (solid bar) cells was quantitated by LC–MS and normalized to total phospholipids of whole cell extracts. Data are shown as mean±S.D. of three different experiments. *P<0.01. (b) Sphk1 (SK1) was overexpressed in SPLhi cells using a pBabe mammalian expression system. SPLhi cells with empty vector (SPLhi-pBabe) or SPLhi cells with forced SK1 expression (SPLhi-SK1) were left untreated or subjected to IR, and 36 h later apoptosis was assessed by measuring caspase-3 activity. Data are shown as mean±S.D. (n=3). (c) Whole cell lysates from SPLhi cells with empty vector (SPLhi-pBabe) or SPLhi cells with forced SK1 expression (SPLhi-SK1) were immunoblotted with SK1 or actin antibodies. (d) Control and SPLhi cells were grown on poly-L-lysine coated culture slides for 24 h. Immunolabeling was performed for ceramide in fixed and permeabilized cells. Images were acquired using a laser-scanning microscope (LSM 710). (e) Fluorescence intensity for ceramide was quantitated using NIH ImageJ software. Data are shown as mean±S.E.M. (n=2, *P<0.01)
