Fig. 6

NNMT expression rescues the GPX8-KO phenotype. A-C, Western blot analysis of total and phosphorylated forms of ACC, AMPK, and NNMT, as normalized by β-actin (A), relative levels of 1MNA, NAD⁺ as measured by LC–MS/MS (B), relative levels of FA de novo synthesis as measured by NMR (C) in WT, GPX8-KO, and GPX8-KO with NNMT overexpression (OE) Caki1 cells. Data from (B) and (C) were normalized by total protein level. Asterisk indicates exogenous NNMT (Myc-tagged NNMT) expression in (A). D, Representative pictures (left) of neutral lipid BODIPY 493/503 from GPX8-KO Caki1 cells with and without NNMT OE. Quantitation of the lipid droplet (right) (n ≥ 3) as in Fig. 3F. E–F, Relative in vitro growth rates (E) and tumor growth (top) and tumor weight (bottom) of in vivo tumor xenograft in nude mice (n = 6–7) (F) for WT, GPX8-KO, and GPX8-KO with NNMT overexpression (OE) Caki1 cells. The experiment was performed three months after that in Fig. 2E and batch effects, i.e., initial lag phases for all the groups, may be present and the absolute values may be different from those in Fig. 2E. Data presented in panels (B), (C), (D), (E), and (F) are means ± SD (n ≥ 3). P-values were determined by unpaired t-test (B), (C), (D), and (F) (bottom) or two-way ANOVA with Geisser–Greenhouse correction (E) and (F) (top). G. Representative pictures of lipid contents (top) (Oil red O staining), GPX8 expression (middle), and NNMT expression (bottom) (IHC staining) from in vivo xenograft tumor tissues in nude mice with WT, GPX8-KO, and GPX8-KO with NNMT overexpression (OE) Caki1 cell transplantation; magnification, X200. For (A) through (G), the WT did not exhibit any appreciable difference from vector control (pCMV6-Entry2- Myc-DDK tag) in terms of GPX8 expression, cell viability, colony formation, and lipid droplet formation (Figs. S7A-D)