Fig. 3: PTGES3 is a regulator of AR.
a, A plot showing proteomic changes upon repression of PTGES3 relative to controls; 22RV1i cells were infected with sgRNAs targeting CTRL (control) or PTGES3 and then subjected to puromycin selection. Cells were collected for label-free shotgun mass spectrometry (n = 3). Volcano plot showing the DE proteins in sgPTGES3 versus sgCTRL groups. b, A plot showing gene expression changes upon repression of PTGES3 relative to controls. LNCaPi cells were infected with sgRNAs targeting CTRL (nontargeting control) or PTGES3. Total RNA was collected for RNA-seq (n = 2 as biological replicates). DE genes between the two groups were highlighted in red in the volcano plot; top ranking DE genes were labeled. AR is not DE when PTGES3 is knocked down. c, C42B, Enzalutamide resistant cell lines MR49F, 22RV1 and VCaP were treated with siRNA targeting control or PTGES3. AR, PTGES3 and GAPDH levels were detected by western blotting. d, LNCaP cells were treated with siRNA targeting control or PTGES3 for 48 h, then stained with PI/Annexin V. The percentage of the apoptotic population was measured by FACS (n = 3 biological replicates; mean ± s.d.). Statistical significance was determined using two-sided t-test (***P < 0.001). e, LNCaP cells were treated with siRNA targeting control or PTGES3. cleaved PARP, PARP, cleaved Caspase-3, Caspase-3 and GAPDH levels were detected by western blotting. f, A diagram showing the potential dual functions of PTGES3 regulating AR. g, Left: diagrams illustrating the wildtype, enzymatic mutation (Y9N) and HSP90 mutation (W106A) isoforms of PTGES3. Right: LNCaP cells stably expressing a TET-ON inducible PTGES3 wildtype, Y9N mutation and W106A mutation were treated with or without 100 ng ml−1 doxycycline. Flag-PTGES3 and GAPDH levels were detected by western blotting. h, LNCaP (TET-ON PTGES3) expressing inducible flag-tagged PTGES3 WT, Y9N mutation or W106A mutation were treated with 100 ng ml−1 doxycycline. Co-IP assays were performed using flag antibody. GAPDH, Flag-PTGES3 and HSP90 amounts were detected by western blotting. i, LNCaPi cells were infected with sgPTGES3-Blast, then treated with DMSO or 100 ng ml−1 doxycycline to overexpress the PTGES3 WT or Y9N or W106A mutant proteins. AR and GAPDH levels were detected by western blotting. Panels f and g created with BioRender.com.
