Extended Data Fig. 4: PTGES3 is required for AR protein levels and AR activity but does not affect AR mRNA levels, mRNA half-life or protein half-life.
a, Analysis of proteomic effects of PTGES3 knockdown versus control for select AR target genes. Mass spectrometry was performed in Fig. 3a and focused results are shown here as a heatmap. b, LNCaPi were infected with sgRNA targeting control or PTGES3. Total RNA was collected for RNA-seq. Gene Set Enrichment Analysis (GSEA) was performed. AR targeted genes are the top rank downregulated gene set. c, sgPTGES3 vs sgCTRL RNA-seq was performed as described in b. The RNA levels of individual Hallmark Androgen response genes were shown in the heatmap. d, LNCaPi were infected with sgRNAs targeting CTRL or PTGES3. AR mRNA levels over 18 s rRNA were measured by real-time PCR (n = 3 as biological replicates; Mean ± SD; ns indicates no significant difference by two-sided t-test analysis). e. LNCaPi cells infected with sgCTRL or sgPTGES3 were treated with 1uM Actinmycin D for 0, 8 or 16 h. RNA was collected to measure the AR mRNA levels using real-time PCR (n = 3 as biological replicates; Mean ± SEM; ns indicates no significant difference by two-way ANOVA analysis). f. LNCaPi cells infected with sgCTRL or sgPTGES3 were treated with protein synthesis inhibitor Cycloheximide (CHX, 5 μM) for 0, 12, 24 h. AR protein levels were detected by western blotting. g, 22RV1 cells were treated with siRNA targeting control or PTGES3. AR, ARV7, and GAPDH levels were detected by western blotting.
