Figure 7. Effect of RIPK3 silencing on edelfosine-induced cell death in U118 cells.

(A) Immunoblot to assess knockdown of RIPK3 protein following transfection with RIPK3-siRNA (100 nM). β-Actin was used as a control for protein loading. Protein expression was evaluated 5 days after transfection. (B) U118 untreated control cells, cells transfected with a non-targeting sequence (siRNA Control), and cells transfected with 100 nM RIPK3-siRNA were incubated with 10 μM edelfosine for 20 h in the absence or presence of z-VAD-fmk, and apoptosis was evaluated by flow cytometry analysis of the cell cycle (sub-G₁ cell population). Data shown are means ± SD of three independent experiments. ***, P<0.001 RIPK3-siRNA+EDLF vs. Control-siRNA+EDLF; ***, P<0.001 z-VAD+RIPK3-siRNA+EDLF vs. RIPK3-siRNA+EDLF, Student's t test. (C) Non-targeting siRNA (control)- and RIPK3-siRNA-transfected cells treated with 10 μM edelfosine were analyzed by cell cycle flow cytometry (sub-G₁ population and percentages of sub-G₁ cells are indicated in each histogram) after 20 h drug treatment (upper panel); brightfield microscopy to visualize cell morphology after 4 h edelfosine treatment (middle panel), and DAPI staining of nuclei following 20 h drug treatment (lower panel). (D) Immunoblotting for cleaved caspase-3 forms and caspase-8 (procaspase-8 and cleaved p18 fragment) in untreated cells (Control), and in control cells, and RIPK3-siRNA-transfected cells treated with edelfosine 10 μM for 9 h. β-Actin was used as a control for protein loading. (E) U118 cells were untreated (C) or treated with necrosulfonamide (NSA) at the indicated concentrations for 21 h, treated with 10 μM edelfosine for 20 h, and pretreated with NSA for 1 h and then treated with edelfosine for 20 h. Then apoptosis was evaluated by flow cytometry analysis of the cell cycle (sub-G₁ cell population). Data shown are means ± SD of three independent experiments. **, P<0.01 and ***, P<0.001 NSA+EDLF vs. EDLF, Student's t test.