Figure 8

Effect of COX-2 inhibition on the cell survival mechanism of v-FLIP/K13-induced anoikis resistance. (A) Expression of (a) cIAP-1, (b) cIAP-2, (c) xIAP, (d) catalase, (e) SOD1, (f) SOD2, (g) BCL2A and (h) IEX-1 in the cells obtained from colonies of untreated, celecoxib or NS-398 solvent-treated and celecoxib or NS-398-treated v-FLIP-HMVECs. (B) Protein levels of Bcl-2, BAX, PARP, cleaved PARP, MCL-1, BCL-xL, BIM and β-actin in the cell lysates prepared from colonies of untreated, celecoxib or NS-398 solvent-treated and celecoxib or NS-398-treated v-FLIP-HMVECs. Blots are representative of three independent experiments. (Ca, b) Celecoxib or NS-398 treatment induces colocalization of BAX and cytochrome c in v-FLIP- HMVECs undergoing anoikis. Untreated and COX-2 inhibitor-treated cells were fixed and immunostained with antibodies against BAX (red) and cytochrome c (green) or with DAPI to detect DNA (blue). Cells were imaged by confocal microscopy. (Da, b) Celecoxib or NS-398 treatment modulates levels of DR4 and DR5 at the protein level. Treated and untreated v-FLIP-HMVECs were stained using DR4 or DR5 antibody. Flow cytometry was performed with a FACS calibur flow cytometer and analyzed with CellQuest Pro software (San Jose, CA, USA). ^* and ^**: statistically significant at P<0.01 and P<0.005, respectively.