Fig 2: Differentiation of mouse CRIPSCs in vivo. The CRIPSCs (P25, 6 months) isolated from TRAMP mice were transplanted into NOG mice for eight weeks, followed by cryosection and immunostaining (A–H). The antibodies included CK8 (A), CK18 (B), AR (C), PSA (D), CK5 (E), p63 (F), Chromogranin A (CHGA) (G), Synaptophysin (SYP) (H). DAPI-stained nuclei. Scale bars, 100 μm

Fig 3: RNF8 promotes the recruitment of AR/ARV7 to the KLK3 promoter.A The lysates from LNCaP cells treated without or with DHT (10−8 M) for 24 h were immunoprecipitated by RNF8 or IgG antibody. Precipitated proteins were detected by western blot. B 22Rv1 cells were maintained in RPMI-1640 with 5% CSS for 24 h, and the cell lysates were immunoprecipitated by indicated antibodies. Precipitated proteins were detected by western blot. C 22Rv1 cells were co-expressed with AR and Flag-RNF8 and treated with DHT (10−8 M) for 24 h. Then, the cells were stained with indicated antibodies. Scale bar, 10 µm. D HEK293 cells with Flag-RNF8 and ARV7 co-transfected were cultured in DMEM with 5% CSS for 24 h, and the cell lysates were immunoprecipitated by anti-Flag M2 resin and IgG. The precipitates were immunoblotted by indicated antibodies. E HEK293 cells were co-transfected with Flag-RNF8 and ARV7 and were maintained in RPMI-1640 with 5% CSS for 24 h. Then, the cells were stained with indicated antibodies. Scale bar, 10 µm. F, G ChIP re-IP was performed in 22Rv1 cells treated without or with DHT (10−8 M) for 24 h using RNF8/AR antibody and IgG as a negative control. The precipitated chromatin was analyzed by the qRT-PCR using primers flanking the KLK3 (PSA) AREI/II. H, I ChIP assays were performed in control or RNF8 knockdown 22Rv1 cells treated without or with DHT (10−8 M) for 24 h by indicated antibodies Data were presented as the fold enrichment relative to IgG control for ChIP re-IP (means ± SEM, n = 3); %input relative to the adjusted input for ChIP (means ± SEM, n = 3). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns no significance (two-sided unpaired t-test).

Fig 4: Confirmation of the role of endogenous Ces1 using CRISPR/Cas9 Ces1‐knockout mice. (A) The results of IHC and HE staining demonstrate the loss of endogenous Ces1 and prostatic intraepithelial neoplasia (PIN) in the anterior prostate of transgenic mice (n = 10 mice per group). Statistical analysis. Representative images are shown. (B and C) Quantification of the corrected total cell fluorescence (CTCF) of BODIPY staining and detection of TG and T‐CHO contents in all groups of prostate tissues were used for generating indicators of intracellular lipids. The graphs show the means ± SEM (n = 10 mice per group), Student's t‐test; *p < 0.05, **p < 0.01, and ***p < 0.001. (D) IHC and TUNEL assays were performed to detect the levels of Ces1, Ki67, Ppara, Perk, caspase‐3, Cyp11a11, and Psa in the Ces1−/− and Ces1+/+ prostate tissues and the apoptosis rate of the Ces1−/− and Ces1+/+ prostate tissue cells. Representative images are shown. Statistical analysis of the IRS scores of IHC staining of CES1‐/‐ or Ces1+/+ prostate tissues. The graphs show the means ± SEM (n = 10 mice per group), Student's t‐test; *p < 0.05, **p < 0.01, and ***p < 0.001. (E) A workflow of the entire study is shown. And, proposed model illustrating the protective function of MLT‐mediated lipid depletion in PCa progression

Fig 5: 6PGD is an androgen receptor (AR)-regulated gene and is elevated in prostate cancer.(A) Effect of siAR and enzalutamide (Enz) on the AR target, PSA. LNCaP cells were transfected with AR (siAR; 12.5 nM) or control (siCon) siRNA for 48 hr or treated with Enz (1 µM) or vehicle (Veh) for 24 hr, after which AR and PSA proteins were evaluated by immunoblotting. GAPDH was used as loading control. (B) Numbers of genes differentially expressed (false discovery rate [FDR] < 0.05) by siAR (versus siCon) or Enz (vs. Veh) are shown in the Venn diagram (at top). Below: an alternative analysis identified 581 genes differentially expressed (FDR < 0.05) by siAR versus Enz. (C) Scatterplot of genes affected by siAR and Enz. The 581 genes differentially expressed by siAR versus Enz are shown in blue (n = 72, genes differentially expressed by siAR versus siCon and Enz versus Veh) and yellow (n = 509), genes differentially expressed by siAR versus siCon but not by Enz versus Veh. (D) Validation of 6PGD expression in response to siAR and Enz by RT-qPCR. Gene expression was normalised to GUSB and L19 and represents the mean ± standard error of the mean (SEM) of three biological replicates; siCon and Veh were set to 1. Differential expression was evaluated using unpaired t tests (a, p<0.01; b, p<0.001; c, p<0.0001; NS, not significant). (E) 6PGD protein levels in response to siAR and Enz treatments were measured by immunoblotting in LNCaP (left) and VCaP (right) cells. HSP90 and GAPDH were used as loading controls. (F) RT-qPCR of 6PGD expression in response to DHT and siAR in VCaP cells. Cells were transfected with siRNAs for 24 hr, and then treated with 1 nM DHT for another 24 hr. Gene expression was normalised and graphed as in (D). Differential expression was evaluated by t tests (**p < 0.01; ***p < 0.001; ****p < 0.0001). (G) RT-qPCR of KLK2, KLK3, and 6PGD expression in response to Enz treatment (1 µM, 72 hr) in patient-derived explants. Gene expression was normalised to GAPDH, PPIA, and TUBA1B and is represented as fold-change relative to vehicle. Differential expression was evaluated by one-sample t tests (**p<0.01; ***p<0.001). (H) 6PGD mRNA expression in prostate tumours pre- and post-androgen deprivation therapy (ADT; GSE48403). A Wilcoxon matched-pairs signed-rank test was used to compare expression in the groups. (I) 6PGD expression is elevated in primary prostate cancer. The TCGA dataset comprises 52 patient-matched normal and cancer samples. Boxes show minimum and maximum (bottom and top lines, respectively) and mean (line within the boxes) values. A paired t test was used to compare expression in normal versus cancer. FPKM: fragments per kilobase of exon per million mapped reads. (J) 6PGD expression by Gleason grade in the TCGA cohort. Boxes show minimum and maximum (bottom and top lines, respectively) and mean (line within the boxes) values. Unpaired t tests were used to compare expression between the groups. (K) 6PGD protein expression in clinical prostate samples (benign prostatic hyperplasia [BPH] and tumours) was measured mass spectrometry. Boxes show minimum and maximum (bottom and top lines, respectively) and mean (line within the boxes) values. An unpaired t test was used to compare expression between the groups. Figure 1—source data 1.Differentially-expressed genes in LNCaP prostate cancer cells treated with siAR or Enz.