Figure 4
From: S1P lyase regulates DNA damage responses through a novel sphingolipid feedback mechanism
ASMase is activated in the presence of SPL. (a) Inhibition of ASMase by desipramine protects against radiosensitivity of SPLhi cells, whereas inhibitors of the de novo pathway do not. Control and SPLhi cells were left untreated or pretreated with fumonisin B1 (25 μM), myriocin (10 μM) or desipramine (20 μM) for 3 h then cells were irradiated. Caspase-3 activity was measured 24 h after radiation exposure. (b and c) ASMase activity is upregulated in SPLhi cells, which was blunted by desipramine. Control and SPLhi cells (1 × 106) were grown in six-well plate for 24 h. SPLhi cells were either left untreated or treated with desipramine (20 μ M) for 6 h. Cells were harvested and then acid (b) and neutral SMase (c) activity was assayed. Data are shown as mean±S.D. (n=4, *P<0.001 (b)). (d) Immunoblotting for ASMase in control and SPLhi cells (left panel). Relative intensity of ASMase expression was quantified by densitometry (right panel). (e) SPL upregulates membrane associated-ASMase. Control and SPLhi cells were grown on poly-L-lysine coated culture slides for 24 h. Immunolabeling was performed for ASMase in fixed, non-permeabilized cells. Images were acquired using a laser scanning microscope (LSM 710). (f) Fluorescence intensity for ASMase was quantitated using NIH ImageJ software. Data are shown as mean±S.E.M. (n=2, *P<0.01)
