Figure 2. Sulindac Induces RXRα-dependent Apoptosis and Bax Activation.

(A–C) The apoptotic effects of Sulindac in F9 or F9 cells lacking RXRα (F9 RXRα^−/−). Cells treated with Sulindac (75 μM) for 24 hr were analyzed by DAPI staining (A), PARP cleavage (B), and DNA fragmentation (C). Scale bar: 10 μm.

(D,E) RXRα siRNA inhibits apoptosis induction by Sulindac. H460 lung cancer cells transfected with control or RXRα siRNA were treated with Sulindac (75 μM) for 24 hr and analyzed by DAPI staining for apoptosis.

(F) Transfection of RXRα enhances the apoptotic effect of Sulindac. CV-1 cells transfected with GFP-RXRα were treated with Sulindac (75 μM) for 24 hr and analyzed by DAPI staining. GFP-RXRα-transfected cells underwent extensive nuclear fragmentation and condensation.

(G) Disruption of the RXRα LBP impairs the apoptotic effect of Sulindac. CV-1 cells transfected with GFP-RXRα or GFP-RXRα/F313S/R316E were treated with Sulindac (75 μM) for 24 hr and analyzed by DAPI staining. Apoptosis scored in receptor-transfected cells. See also Figure S2.

(H,I) Role of Bax in apoptosis induction by Sulindac. HCT116 cells or HCT116 cells lacking Bax (Bax^−/−) were treated with or without sulindac (75 μM) for 24 hr. Apoptosis determined by PARP cleavage (H) and DAPI staining (I).

(J,K) RXRα siRNA inhibits sulindac-induced Bax activation. Knocking down RXRα in HCT116 cells by RXRα siRNA revealed by immunoblotting. HCT116 cells transfected with or without RXRα siRNA or control siRNA for 48 hr were treated with Sulindac for 6 hr, and analyzed for Bax oligomerization (J) and Bax conformational change and mitochondrial targeting by immunostaining/confocal microscopy using Bax/Δ21, Bax/6A7, or anti-Hsp60 antibody (K). About 60% of cells showed Bax conformational change presented.

One of three to five similar experiments is shown. Error bars represent SEM.