Skip to main content
. 2024 May 14;134(13):e171294. doi: 10.1172/JCI171294

Figure 2. l-2HG suppression of ATF4.

Figure 2

(A) Immunoblot for ATF4 protein from 786-O, OS-RC-2, and 769p RCC cells stably transduced with the indicated vectors. Actin was used as loading control. (B) Immunoblot for ATF4 protein from 786-O cells stably transduced with control vector, L2HGDH (WT), or L2HGDH A241G (mutant). Actin was used as loading control. (C) Immunoblot for L2HGDH and ATF4 protein from control and L2HGDH-KO HK-2 cells. Actin was used as loading control. (D) Immunoblot for ATF4 and PHGDH from 769p cells stably expressing either control vector or ATF4 cDNA. Actin was used as loading control. (E) Relative ATF4 mRNA normalized to TBP was examined by RT-qPCR from 769p and 786-O cells stably expressing either control (black) vector or L2HGDH cDNA (red). Data are expressed as mean ± SD from n = 3 (769p) or n = 4 (786-O) biological replicates. *P < 0.05, **P < 0.005. (F and G) 786-O (F) and A498 (G) cells stably transduced with L2HGDH cDNA were transiently transfected (55 hours) with either scramble siRNA (Scr) or siRNAs targeting KDM4C (#1, #2). Immunoblotting for KDM4C, ATF4, PHGDH, and PSAT1 was performed. Actin (β-actin) or tubulin (α-tubulin) was used as loading control. (H) Top: The construct containing the human ATF4 5′-uORFs preceding firefly luciferase. Bottom: Relative luciferase signal following transient transfection of the luciferase construct into 769p and 786-O cells stably expressing the indicated vector (control vector, black; L2HGDH WT, red; or L2HGDH A141G mutant, blue). Data were normalized to Renilla luciferase (Luc). Data are presented as mean ± SEM. ANOVA was used, and Tukey’s post hoc P values are shown.