Figure 1. High l-2HG suppresses amino acid synthesis and transporter genes in RCC.

(A) Differentially expressed transcripts in RXF-393 control vector (high l-2HG) relative to cells transduced with L2HGDH cDNA (low l-2HG). Amino acid synthesis and transporter genes are indicated (horizontal dashed line denotes P value of 0.05). (B) Relative mRNA of PHGDH and PSAT1 (normalized to RPLPO) from RXF-393 cells stably expressing control black vector or L2HGDH (red). Data are shown as mean ± SD from n = 4 biological replicates. (C and D) Relative mRNA levels of amino acid synthetic/transporter genes from 769p (C) and 786-O (D) cells stably expressing the indicated construct. Expression was normalized to RPLPO. Data are shown as mean ± SD from n = 4 biological replicates. *P < 0.05, **P < 0.005, ***P < 0.0001. (E) Immunoblot of PHGDH, PSAT1, and L2HGDH protein from 769p and 786-O cells transduced with control vector or L2HGDH cDNA. Actin (β-actin) or Ponceau S stain was used as loading control. Blots are from the same biological sample run contemporaneously. (F) Immunoblot of ASNS protein from 786-O and OS-RC-2 RCC cells transduced with control vector or L2HGDH cDNA. Actin was used as loading control. (G and H) Immunoblot of RXF-393 (G) and 769p (H) cells stably expressing control vector, L2HGDH (WT), or L2HGDH A241G (catalytic mutant). Actin was used as loading control. (I) Tandem MS analysis for l-2HG and D-2HG metabolites from control (black) or L2HGDH-KO (red) HK-2 renal epithelial cells normalized to protein content. Data are shown as mean ± SEM from n = 3 biological replicates. (J) mRNA expression of the indicated genes was examined by RT-qPCR from control (black) or L2H DH-KO (red) HK-2 cells. Data are expressed as mean ± SEM from n = 3 biological replicates. *P < 0.05, **P < 0.005. (K) Immunoblot for PHGDH protein from HK-2 cells treated with either DMSO or l-2HG octyl ester (5 mM) or DMOG (1 mM) for 4 hours. Actin was used as loading control.