Extended Data Fig. 6: BCAA catabolism potentiates PCa progression.
a. Growth curves of PCa cells with or without BCAT2 KD (n = 7 biological replicates for each group; Data represent the mean ± SEM, the P value was determined by two-way ANOVA followed by multiple comparisons). b. IB analysis of BCAT2 expression in PCa cells. Representative result of three biological replicates is shown. c. Growth curves of LNCaP cells with or without BCAT2 overexpression (n = 7 biological replicates per group; Data represent the mean ± SEM, the P value was determined by two-way ANOVA followed by multiple comparisons). d. IB analysis of BCAT2 in PCa cells as indicated. Representative result of three biological replicates is shown. e. Representative images and quantification of formation efficiency and size of PtenPC−/− organoids with or without BCAT2 overexpression (n = 10 biological replicates for each group, data represent the mean ± SEM; the P value was determined by two-tailed t-test). Scale bar, 500 μm. f-g. Representative image and quantification of the formation efficiency and size of PtenPC−/−; Tp53PC−/− organoids with or without Bcat2 knockdown (n = 15 biological replicates per group, data represent the mean ± SEM; the P value was determined by one-way ANOVA followed by multiple comparisons test). 500 μm. h. H&E and IHC staining for Ki67 in prostate sections from the orthotopic transplantation of PtenPC−/−; Tp53PC−/− organoids and quantification of Ki67+ cells (n = 10 tumors for each group). Data represent the mean ± SEM, the P value was determined by one-way ANOVA followed by multiple comparisons test. Scale bar, 100 μm. i. Relative levels of acetyl-CoA in PtenPC−/−; Tp53PC−/− organoids with or without Bcat2 knockdown (n = 3 biological replicates per group). Data represent the mean ± SEM, the P value was determined by one-way ANOVA followed by multiple comparisons test. j-k. BCAT2 knockdown suppresses mitochondrial respiration levels (OCR) in 22RV-1 (n = 5 biological replicates per group) and PtenPC−/−; Tp53PC−/− organoids (n = 3 biological replicates per group, data represent the mean ± SEM; the P value was determined by two-way ANOVA followed by multiple comparisons test). l. The glycolytic flux (ECAR) analysis in PtenPC−/−; Tp53PC−/− organoids with or without Bcat2 deletion (n = 3 biological replicates for each group). Data represent the mean ± SEM, the P value was determined by two-way ANOVA followed by multiple comparisons test. m. Measurement of 22RV-1 growth in WT and BCAT2-depleted cells with or without nucleobases (NB) and/or BCKA supplementation. Data represent the mean ± SEM; P value was determined by two-way ANOVA followed by multiple comparisons test. (n = 5 biological replicates per group), no significance. n. Measurement of PtenPC−/−; Trp53PC−/− organoids size in WT and BCAT2-depleted cells with or without nucleobases (NB) and/or BCKA supplementation. Data represent the mean ± SEM; P value was determined by one-way ANOVA followed by multiple comparisons test. (n = 16 biological replicates per group). NS, no significance.
