Involvement of AIF release from the mitochondria in the antitumor effect of celecoxib or E7123 in DLBCL cell lines. (A) Immunoblot analyses of AIF, Omi/HtrA2, and EndoG in the cytosolic fraction extracts after incubating HT, DB, and KARPAS-422 cells with 60μM of compound or vehicle for 1 or 4 hours. The remaining extract containing mitochondrial and nuclear fractions of vehicle-treated cells was used as a control for the presence of the proteins in the mitochondria. The absence of COX-II expression indicated no mitochondrial contamination of the cytoplasmic fraction. GAPDH was used as a cytoplasmic fraction marker as well as a control for protein loading. (B) Immunolocalization of AIF after cell exposure to celecoxib (CX) or E7123. HT and KARPAS-422 cells were treated with vehicle, 60μM celecoxib for 4 hours, or 60μM E7123 for 1 hour. AIF was stained with anti–AIF-tetramethylrhodamine isothiocyanate antibody (red, second column), the nucleus was stained with Hoechst (green, first column), and colocalization was detected by merging (yellow, third column; original magnification ×630). Slides were viewed with an inverted microscope (Axiovert 200M). Images were acquired using a digital camera (Coolsnap; Photometrics) and processed with MetaMorph Version 5.01 software. (C) Cytotoxic effect of 60μM celecoxib or 30μM E7123 after 4-hour exposure in DB, HT, KARPAS-422 cells pretreated with 10μM of CsA, an MPT inhibitor, for 30 minutes. Error bars represent SE. Statistical analysis was performed using the Mann-Whitney test: *P < .05; **P < .01.