Figure 5
Indomethacin downregulates Wnt/β-catenin signaling in platinum-resistant ovarian tumor cells. (a) Western blot showing robust decrease in β-catenin protein level by 24 h upon treatment with Indomethacin in OV81.2-CP10 and CP70 cells and enhanced β-catenin downregulation in cisplatin plus Indomethacin combination treated OV81.2-CP10 and CP70 cells as compared with individual drug treatment alone 24 h. (b) Real-time PCR analysis 24 h showing Indomethacin decreases mRNA expression of β-catenin transcriptional targets AXIN2, TCF7, LEF-1 and LGR5 in OV81.2-CP10 and CP70. (c) Western blot showing overexpression of non-degradable β-catenin (β-S33Y) in OV81.2 (left) and effect of Indomethacin on β-catenin protein expression in OV81.2-β- S33Y cells (right). (d) clonogenics assay on day 7 showing β-S33Y overexpression partially rescues the effect of Indomethacin on cell survival in OV81.2. (e) 48 h Annexin-V PI staining flow cytometry analysis showing that β-S33Y overexpressing OV81.2 cells are more tolerant to Indomethacin induced cell death. (f) Western blot confirming lentiviral shRNA mediated β-catenin knockdown. (g) 48 h analysis of Annexin-V PI staining showing increased cell death upon Indomethacin treatment in OV81.2-CP10 and CP70 sh-β-catenin cells compared with sh-scram control (*P<0.05, **P<0.005 and. ***P<0.0005).
