Journal: Cell Death & Disease
Article Title: Integrated analysis reveals FOXA1 and Ku70/Ku80 as targets of ivermectin in prostate cancer
doi: 10.1038/s41419-022-05182-0
Figure Lengend Snippet: A GSEA showed that genes induced by FOXA1 were inhibited by ivermectin in C4-2 cells. B RT-qPCR analysis of FOXA1 induced genes ( CDKN3 , CDCA2 , and CAMKK2 ) and FOXA1 repressed EMT-associated genes ( MET , MMP7 , and SOX9 ) in C4-2 cells treated with ivermectin for 48 h. C Western blot analysis of FOXA1 and N-cadherin in LNCaP and C4-2 cells treated with ivermectin for 48 h. D ChIP–qPCR analysis for FOXA1 or AR occupancy, and FAIRE–qPCR analysis of chromatin accessibility at a target regulated by AR and FOXA1 (KLK3 and NKX3-1) in C4-2 cells treated with ivermectin. E ChIP–qPCR analysis for FOXA1 and FAIRE-PCR analysis of chromatin accessibility at a target regulated by FOXA1 (E2F1 and MET) in C4-2 cells treated with ivermectin. F FOXA1 knockdown impaired the ivermectin-repressed expression of KLK3 and E2F1 genes. mRNA levels were measured 48 h after the implementation of the ivermectin treatment and siRNA transfection by RT-qPCR in C4-2 cells. G , H Western blots showing thermostable FOXA1 and AR following indicated heat shocks in the presence (+) or absence (−) of 50 μM ivermectin in LNCaP ( G ) and C4-2 ( H ) cells. I Western blots showing thermostable FOXA1 following indicated heat shocks in the presence (+) or absence (−) of 50 μM ivermectin in 22RV1 cells. J GSEA showed the inactivation of FOXA1 induced genes in 22RV1 cells after the ivermectin treatment. K RT-qPCR analysis of FL-AR and ARv7 in 22RV1 cells treated with ivermectin for 48 h. L ChIP–qPCR analysis for FOXA1 and FAIRE–qPCR analysis of chromatin accessibility at KLK3 and E2F1 in 22RV1 cells treated with ivermectin.
Article Snippet: The samples were analyzed by immunoblotting with primary antibodies to: PARP (Cell Signaling Technology Cat# 9532, 1:1000), cleaved-caspase 3 (Cell Signaling Technology, Cat# 9664, 1:1000), γH2A.X (Cell Signaling Technology Cat# 2577, 1:1000), AR (Santa Cruz Biotechnology Cat# sc-7305, 1:1000), PSA (Cell Signaling Technology Cat# 5365, 1:1000), UBE2C (Cell Signaling Technology Cat# 14234, 1:200), E2F1 (Cell Signaling Technology Cat# 3742, 1:1000), FOXA1 (Cell Signaling Technology Cat# 53528, 1:1000), Ku70 (Cell Signaling Technology Cat# 4588, 1:1000), Ku80 (Cell Signaling Technology Cat# 2180, 1:1000), BRCA1 (Cell Signaling Technology Cat# 9009, 1:1000), Rad51 (Cell Signaling Technology Cat# 8875, 1:1000), Lamin B (Cell Signaling Technology Cat# 13435, 1:1000), and GAPDH (Santa Cruz Biotechnology Cat# sc-47724, 1:1000).
Techniques: Quantitative RT-PCR, Western Blot, Expressing, Transfection