Fig. 3

The androgen deprivation upregulates PHB1 expression and alters its subcellular distribution. A. The mRNA and protein levels of PHB1 in LNCaP cells were determined by qRT-PCR and Western blot. LNCaP cells were treated with 1 nM DHT at the indicated time points (left two panels) or treated with 0.01, 0.1, 1, and 10 nM DHT for 24 h (right two panels). Western blot was performed with the indicated antibodies. For qRT-PCR, β-actin was used as the reference gene. For Western blot, β-tubulin was used as a loading control. **P < 0.01, ***P < 0.001. d, days. h, hours. CSS, charcoal-stripped serum. B The mRNA and protein levels of PHB1 in LNCaP cells following chronic androgen deprivation were analyzed by public dataset and Western blot. Left: mRNA levels of PHB1 in public dataset (GSE59986); Right: protein levels of PHB1 in LNCaP and LNCaP-AI cells. Western blot was performed with the indicated antibodies. β-tubulin was used as a loading control. d, days. w, weeks. m, months. LNCaP cells were cultured under CSS condition for 3 d, then, were used for the following series of experiments (C-H). C Protein levels and subcellular distribution of PHB1 was determined by Western blot and subcellular fractionation. Left: total expression; Middle: nuclear (N) and cytosol (C) expression; Right: plasma membrane 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. D 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). E 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. F Binding of AR to the ARE enhancer regions of TMPRSS2 (above) and PSA (below) genes were monitored by ChIP assay. Cell lysates were immunoprecipitated with anti-AR. Purified rabbit IgG was used as a negative control. **P < 0.01, ***P < 0.001. G Phosphorylation level of c-Raf^Ser338 and MEK with transfection of siNC/siPHB1 was determined by Western blot. Western blot was performed with the indicated antibodies. β-tubulin was used as a loading control. Densitometry analysis was performed using ImageJ, with target protein bands normalized to β-tubulin bands. Representative images are shown. H 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 upper/bottom right panel. Colocalization signal is yellow (black arrows). FBS Fetal bovine serum. CSS Charcoal-stripped serum. d Days