RIP1 is required for CD40-mediated apoptosis in carcinoma cells. (A) Knockdown efficiency of RIP1 siRNA. (B) EJ cells treated with CD154 in the presence of CHX display morphology typical of apoptotic cells (see representative arrowheads), whereas RIP1 siRNA-transfected cultures remain unaffected. Bar, 25 µm. (C and D) RIP1 knockdown rescues EJ cells from CD154 and CHX-induced apoptosis, as determined by propidium iodide staining and immunofluorescence microscopy (C) or cell death ELISA (D). Mean values (±SD) from four independent experiments are shown. (E and F) RIP1 knockdown rescues HeLa/CD40mT6 cells from CD154-induced apoptosis, determined as described in the legend of Fig 4 C. (G and H) Expression of cIAP1/2 (G) and CYLD (H) before and after transfection with the respective siRNAs. (I) Effect of cIAP1/2 and CYLD knockdown on CD154-mediated EJ cell death. (J) Necrostatin (50 µM) inhibits and the pan-caspase inhibitor zVAD-fmk (15 µM) abolishes the pro-apoptotic effects of CD154 in EJ cells. Error bars indicate SD.