Fig. 5
From: Targeting PHB1 to inhibit castration-resistant prostate cancer progression in vitro and in vivo
FL3 inhibits ENZ-sensitive CRPC by affecting the subcellular distribution of PHB1. C4-2B cells were treated with DMSO or FL3 20 nM/40 nM for 48 h. A The effects of FL3 on PHB1 expression and its subcellular distribution was determined by Western blot and subcellular fractionation. Left: total expression; Middle: nuclear (N) and cytosol (C) expression; Right: plasma membranes expression. Western blot was performed with the indicated antibodies. PHB1 bands were normalized to β-tubulin bands (total and cytosol expression)/Lamin A/C bands (nuclear expression)/ Na+-K+-ATPase bands (plasma membrane expression). Representative images are shown. B Subcellular distribution of PHB1 and AR was analyzed by IF assay. Representative images are shown with a 5 µm scale-bar. PHB1: green; AR: red; DAPI: blue. Magnified images from the regions marked by rectangles showed in the bottom right panel. Colocalization signal is yellow (black arrows). C PHB1-AR interaction in whole cell lysate (left) and nuclear extract (right) was determined by Co-IP assay. Cell lysates were immunoprecipitated with anti-PHB1. IgG serves as negative control. D Binding of AR/PHB1 to the ARE enhancer regions of TMPRSS2 (above) and PSA (below) genes were monitored by ChIP assay. Cell lysates were immunoprecipitated with anti-AR or anti-PHB1. Purified rabbit IgG was used as a negative control. *P < 0.05, **P < 0.01. E mRNA levels of PHB1, AR, PSA, and TMPRSS2 was determined by qRT-PCR. β-actin was used as the reference gene. **P < 0.01. F Subcellular distribution of PHB1 and mitochondria were analyzed by IF assay. Representative images are shown with a 5 µm scale-bar. PHB1: green; MitoTracker: red; DAPI: blue. Magnified images from the regions marked by rectangles showed in the bottom right panel. Colocalization signal is yellow (black arrows). G. Apoptosis rate of cells was determined by Flow cytometry assay. Representative bar diagram of three independent experiments is presented. **P < 0.01, ***P < 0.001. H. The EGF-induced phosphorylation levels of c-RafSer338 and MEK in cells with siNC/siPHB1 transfection were determined by Western blot. Western blot was performed with the indicated antibodies. β-tubulin was used as a loading control