A. Total cell lysates (20µg) from the indicated prostate cancer cells were analyzed by western blots using specific antibodies against Drp1, androgen receptor (AR), PSA, COXIV and GAPDH. B. The above blots were scanned and the densitometric values for Drp1 and AR were normalized to GAPDH and plotted as a percent of the values. The values for the cell line CWR-R1, which express the highest levels of Drp1 and AR were set at 100% and a correlation coefficient was calculated based on these values using GraphPad Prism software. C. Total RNA from prostate cancer cells was isolated, cDNA was prepared and Drp1 mRNA levels were analyzed by qRT-PCR. Drp1 mRNA levels were normalized to GAPDH and the data expressed relative to mRNA levels of normal prostate epithelial cells, P69. D. Drp1 expression in prostate cancer (n=7) and hormone refractory metastatic prostate cancer (n=6) were analyzed by Western blot. Expression of PSA served as an indicator of androgen responsiveness, while GAPDH was used as loading control.

A and B. LNCaP cells were treated with varying concentrations of bicalutamide (BIC) in the presence of R1881 (10nM), and total cell lysates were analyzed by western blotting (A) or total RNA was analyzed by qRT-PCR for Drp1 mRNA levels (B). For qRT-PCR results represent the means ± SEM of three separate experiments with *P<0.01, **p<0.001 relative to R1881 treatment in the absence of bicalutamide. C. LNCaP cells were transfected either with siRNA against AR or scrambled siRNA. Total cell lysates were analyzed for the expression of Drp1, AR and β-actin (loading control). D. cells were treated with R1881 (10nM) for 8h or 12h, either in the presence or absence of the protein synthesis inhibitor cycloheximide (CHX; 50µg/ml, 1h before and during R1881 treatment). Drp1 mRNA levels were analyzed by qRT- PCR (left). Total cell lysates were analyzed for the expression of Drp1 (right). The results represent the means ± SEM of three separate experiments.

A. Androgen-sensitive VCaP cells were treated with R1881 and bicalutamide (BIC), and total cell lysates were analyzed by western blot for Drp1 expression. B. Androgen depletion-independent C4-2 cells were treated and analyzed as in (A). C & D. Androgen refractory, AR-negative DU145 (C) and PC3 cells (D) were treated with R1881 (10nM) for 24h. All cells were grown in steroid-depleted medium for 48h before R1881 treatment and analyzed as in (A).

A. In the left panel, cells were grown in medium containing 9% FBS or charcoal/dextran-stripped serum (CSS); and in the right panel, cells were treated with R1881 (10nM) for 24h. Total cell lysates were analyzed by western blot for the levels of Drp1, PSA and AR. Hsp90α was used as a loading control. B. Total RNA was isolated and Drp1 (left) and PSA (right) mRNA were measured by qRT-PCR (**P≤0.01; ***P≤0.001, N=3). C. Cells were treated with increasing concentrations of R1881 for 24h and total cell lysates were analyzed for the levels of Drp1 protein. D. Cells were treated with R1881 for different periods and Drp1 mRNA expression was analyzed by qRT-PCR (P values were compared to the CSS controls, N=3).

A. AR ChIP-seq data analysis showing .wig data of an AR binding site located in the Drp1 gene. As denoted by 'KLK2' in the figure, the sequence in red denotes only AR binding sites that are as enriched or more enriched than the binding site in kallikrein 2 (KLK2), a prostate-associated protease that activates PSA. The data predict an AR binding site that is downstream of the RNA PolII site at the start of the Drp1 (also known as dynamin-like protein 1, or DNM1L) gene, located at chr12:32,724,000–32,725,500. B. LNCaP cells were treated with R1881 for different time periods and isolated mRNA was subjected to Illumina BeadArray analysis. The two plots represent results obtained with two separate probes for Drp1. TMPRSS2 was analyzed as a positive control for an androgen-responsive gene. Open circles represent cells treated with androgen and blue circles with vehicle (EtOH).

Stably transfected mito-green LNCaP cells were treated with R1881 (1nM) for 24h with or without CGP (50µM) for the last 1h, the cells were washed with HBSS and live cells were observed in a confocal fluorescent microscope. A. A representative photograph of cells in each treatment group is shown, illustrating the shape of the mitochondria. These pictures are from confocal imaging (Zen software) using ‘Z-stack’ option and signals from all the planes were considered by using ‘Maximum Intensity Projection’ option. B. Cells exhibiting more than 80% punctate (fragmented) mitochondria were counted and presented as a percentage of the total number of cells counted. At least 200 cells were examined in each dish and values represent the means ± SEM from at least 3 separate experiments, with **P≤0.01; *** P≤0.00 relative to the control.

A. Androgen increased cell proliferation. LNCaP cells were treated with R1881 (1nM), bicalutamide (BIC, 50µM) or CGP (50µM) as described above. Cell proliferation was measured using a Quick Cell Proliferation assay kit (BioVision). Data are expressed as means ± SEM, with the brackets indicating the P values (N=5) (left panel). Right Panel: Cells were treated with R1881 (1nM) for 48h, fixed overnight with 70% ethanol, treated with RNAse and then stained with propidium iodide and analyzed using flow cytometry to determine the S-phase of the cell cycle (***P≤0.0002; N=3). B. Androgens increased phosphorylation of Drp1-Ser-616. LNCaP cells were treated with R1881 (1nM) and CGP (50µM for 18h) as described earlier. Cell lysates were subjected to western blot analysis and Drp1 phosphorylation at Ser-616 and total Drp1 determined. The lower panel shows the densitometric values for phospho-Ser-616 Drp1 levels normalized to the levels of total Drp1 in the above blot. C. Androgens increased CGP-induced apoptosis. LNCaP cells were treated with R1881 (1nM) for 24h. Cells were treated with CGP (50µM) for an additional 4h or 18h in the presence or absence of R1881. Cell lysates were used for an apoptosis assay using an M30 apoptosense analyses kit. Results are presented as the percentage of apoptosis compared to the CSS control (*P≤0.05; **P≤0.01; N=3). D. DN-Drp1 reduced androgen-mediated CGP-induced apoptosis. LNCaP cells were transfected with DN-Drp1 and treated with R1881 (1nM) and CGP (50µM for 18h) as described earlier. Cell lysates were analyzed for apoptosis using an M30 apoptosense analysis kit. Results are presented as the percentage of the respective controls (**P≤0.01; N=3). E. CGP induced a decrease in Mfn1. LNCaP cells were treated as in (B) and total cell lysates were analyzed for Mfn1 and Mfn2 levels.