Fig. 4: HMMR inhibition decreases AR nuclear translocation and transcriptional activity.

a Decreased nuclear localisation of pCMV-tagged wtAR (green) in transfected PC3 cells treated with 4-MU or docetaxel (DTX) ± dihydrotestosterone (DHT), captured by fluorescence microscopy. Nuclei were visualised with DAPI mounting media (blue). Data are presented as the mean ± SD of 2 wells and represent two independent experiments. Data were analysed using two-way ANOVA with Tukey’s multiple comparison test. (***p < 0.001, ****p < 0.0001). The scale bar represents 10 pixels/micron. b Western Blot of AR protein expression in LNCaP nuclear fraction upon treatment with vehicle, 10 nM DHT, 0.4 mM 4-MU or DHT + 4-MU. Acetylated Histone H3 was used as a nuclear marker. Numerals above each lane represent densitometric analysis of AR relative to H3. c LNCaP cells were androgen deprived for 3 days prior to treatment with DHT ± 4-MU. Cells were analysed by RT-qPCR (upper panel) or Western Blot (lower panel) for KLK3 expression. Genes were normalised to GUSB and L19. Loading control for Western Blot was GAPDH. PCa cells grown in the presence of androgens were treated with increasing doses of 4-MU. Cells were analysed by RT-qPCR (upper panel) or Western Blot (lower panel) for KLK3 expression in LNCaP (d), V16D (e) and MR49F (f) cells. Genes were normalised to GUSB and L19. Loading control for Western Blots was β-Actin. Numerals above each lane represent densitometric analysis of each protein relative to β-Actin. Data in c–f are presented as mean ± SD of 3 wells and analysed using one-way ANOVA with Dunnett’s test (***p < 0.001, ****p < 0.0001). g HMMR, AR and PSA protein expression after 72 h of siHMMR knockdown. Numerals above each lane represent densitometric analysis of each protein relative to the loading control β-Actin. Data represents two independent experiments. RT-qPCR of KLK3 expression in response to 72 h of siHMMR knockdown in LNCaP (g) and MR49F (h) cells. Genes were normalised to GUSB and L19. Data are presented as mean ± SD of three biological replicates and two independent experiments. siControl was set to one and data was analysed by unpaired student’s T-test (siHMMR vs siCON; **p < 0.01, ***p < 0.001).