Fig. 4. AA inhibits CRPC tumour growth in vivo.

C4-2 cells were injected in each flank of immune-deficient mice. Tumour growth, tumour proliferation and mouse organs were analysed. a Tumour volume in AA-treated mice compared to control. Male nude mice received subcutaneous inoculation with C4-2 cells in each flank. Upon tumour formation mice were randomly grouped (control n = 6, AA n = 7) and injected daily with vehicle or AA (100 mg/kg). Tumour volume was measured every 3 days using a calliper. Two-way ANOVA test was performed. *p < 0.05, t = 2.790. b Body weight of mice was measured once a week and plotted against days of treatment. c The weight of Levator ani from control compared to AA-treated mice. d Representative images of immunofluorescence staining of mouse prostates. Mouse organs were isolated from mice sacrificed at day 19. Bright-field (BF) and scale bars indicate 100 μm. Relative level of Ki67 expression per cell nucleus was analysed and noted as relative units (r.U.). 20 images were analysed per tissue and treatment; n = 5 per treatment group, p = 0.80. e qRT-PCR analyses of various prostate-specific AR target genes for murine prostate with seminal vesicles of xenografted mice (control n = 5, AA n = 6). Rpl13a was used as housekeeping gene for normalisation. f Different organs from xenografted mice (control n = 5, AA n = 6) were analysed for organ-specific AR target gene expression by qRT-PCR. β-Actin was used as housekeeping gene for normalisation. Mann–Whitney U test was used with error bars indicating SEM. g Western blot analysis of PSA protein levels in the xenografted tumour tissues isolated from vehicle- or AA-treated mice (n = 6). The normalised protein levels are indicated along with the values underneath the brackets. h qRT-PCR results of extracted RNA from tumour tissues isolated from xenografts to analyse expression of AR target genes. Both β-Actin and RPL13a were used as housekeeping genes for normalisation. Mann-Whitney U test was used with error bars indicating SEM.