Figure 4

Reagents inhibiting shRNA target proteins identified in the screen suppress FASL/TNF-induced cell death in vitro. (a) Known pharmacological inhibitors of non-canonical targets identified in the library screen were tested for their effect on survival of cultured cells exposed to FASL or TNF. The tested inhibitors included Bim23056 (an SSTR5-blocking peptide, left) and cantharidin (a small-molecule PPP2R inhibitor, right). HeLa cells were pretreated for 12 h with the indicated concentrations of inhibitors and then treated with FASL (50 ng/ml, solid blue line) or TNF (50 ng/ml, blue dashed line) in the presence of cycloheximide (CHX, 2.5 mg/ml). The red lines correspond to treatment of cells with inhibitors alone (without FASL or CHX), plotted on the red y-axis on the left. Treatment with inhibitors and FAS/CHX is plotted on the blue y-axis on the right. Cell viability was determined 24 h after FASL addition by quantitation of methylene blue staining (optical density (OD) at 540 nm). Error bars indicate standard deviations for the means of three independent experiments. (b–d) Chemical inhibition of candidate targets SSTR5 and PPP2R5A leads to suppression of TNF- and FASL-induced activation of caspases 3/7 and 8 in vitro. (b) Caspase-3/7 activity was determined by Caspase-Glo assay (a luminogenic assay with a DEVD-containing substrate; RFU, relative fluorescence units) in A549 (top), H1299 (middle) and HeLa (bottom) cells left untreated, treated with TNF (50 ng/ml)+cycloheximide (2.5 μg/ml), Bim23056 (200 μM) or cantharidin (0.8 μM) alone, or pretreated with Bim23056 (200 μM) or cantharidin (0.8 μM) for 0.5 h before addition of TNF+cycloheximide. Caspase activity was determined 4 h after TNF application. (c) Caspase-3/7 activity was determined in HeLa cells as described in b except that FASL (50 ng/ml) was used instead of TNF and caspase activity was measured at 6 h after FAS application. (d) Caspase-8 activity was determined in A20 cells as in c with the only difference being use of an LETD-containing luminogenic substrate. For (b–d), the values shown are the means of at least three independent experiments. Error bars indicate standard deviations. P-values were calculated using a moderated Student’s t-test with FDR correction