Fig. 5

NNMT mediates GPX8’s inhibition of AMPK. A, Correlation between GPX8 and NNMT mRNA expression in ccRCC patients from TCGA-KIRC dataset. B, Volcano plot for NNMT mRNA expression from RNA-seq data comparing GPX8-KO vs. WT Caki1. C, NNMT expression in different subtypes of renal cancer and normal tissues from TCGA-KIRC. P-values were determined by Mann–Whitney U test. D, Western blot analysis of NNMT in GPX8-KO Caki1 and shGPX8 786O vs. control cells. E, Relative levels of 1MNA and NAD ⁺ from GPX8-KO and WT cells as measured by LC–MS/MS. F, Western blot analysis of total and phosphorylated forms of ACC (Ser 79) and AMPK α1 (T183) α2 (T172) in Caki1 and 786O upon NR treatment for 2 days. G, Relative levels of NAD ⁺ between tumor and matched normal tissues in ccRCC patients from previous dataset [35]. P-value was determined by Wilcoxon matched–paired signed-rank test. H–L, The effect of shNNMT in Caki1 and 786O cells. Relative levels of 1MNA and NAD ⁺ as measured by LC–MS/MS (H), western blot analysis of total and phosphorylated forms of ACC (Ser 79) and AMPK α1 (T183) α2 (T172) (I), FA de novo synthesis (CH3ω) and triacylglycerol synthesis (TG) (J), representative pictures (top) of neutral lipid BODIPY 493/503 staining from Caki1 shNNMT and 786O shNNMT with quantitation of the lipid droplet (bottom) (n = 3) as in Fig. 3F (K), and relative growth rates (L). P-value was calculated by two-way ANOVA with Geisser–Greenhouse correction (L). Western blot analysis was normalized by β-actin. Data from (E), (H), and (J) are normalized by total protein level. Data from (E), (H), (J), and (K) are means ± SD (n ≥ 3). P-values were determined by unpaired t-test