Figure 1
From: S1P lyase regulates DNA damage responses through a novel sphingolipid feedback mechanism
SPL is a radioresponsive protein that increases radiation-induced cell death. (a) HEK293T and NIH3T3 cells were left untreated or irradiated with 10 Gy of X-rays. Cells were harvested 9 h (HEK293T) or 24 h (NIH3T3) after radiation exposure and whole cell lysate was immunoblotted with actin and anti-human SPL, or anti-murine SPL antibodies, respectively. (b and c) Control and SPLhi cells were exposed with indicated dose of IR. After 24 h of incubation, cells were harvested, and apoptosis was assessed by measuring caspase-3 activity (b) and cell lysate was immunoblotted for PARP (Poly (ADP-ribose) polymerase). (c) Caspase-3 activity experiment represents mean±S.D. of three independent experiments. The immunoblot is representative of at least three independent experiments. (d) Control and SPLhi cells were left untreated or subjected to 5 Gy dose of X-rays and cells were harvested 12, 24 and 36 h after radiation exposure. Apoptosis was determined by flow cytometry using propidium iodide staining. Values are shown as mean±S.D. of three independent experiments. (e) Control, SPLhi, and SPLhi cells expressing a shRNA specific for Sgpl1 (SPLhi/shRNA) were exposed to 10 Gy IR. After 24 h of incubation, caspase-3 activity was measured in whole cell lysate. Sgpl1 knockdown was confirmed by western blotting (inset). Data represents mean±S.D. of three different experiments done in duplicates. (f) Endogenous knockdown of Sgpl1 induces radio-resistance. HEK293T control cells with lentiviral vector (control, open bar) or with shRNA specific for sgpl1 (SPLlo; solid bar) were exposed to 10 Gy X-rays and caspase-3 activity was measured 36 h after radiation exposure. Sgpl1 knockdown was confirmed by RT-PCR (inset). Data are shown as mean±S.D. (n=3, *P<0.01). Each experiment was repeated at least three times. Data points indicate average±S.D. of representative experiment
