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. 2023 Apr 24;42(12):e112675. doi: 10.15252/embj.2022112675

Figure EV1. GDH1 depletion sensitizes CRC cells to hypoxia.

Figure EV1

  • A
    Construction of GDH1 depleted cell lines.
  • B
    Cell proliferation assay was performed using cell lines constructed in Fig S1A (n = 3).
  • C, D
    HCT116 and SW480 cells with or without GDH1 were supplemented with cell permeable methyl‐αKG under hypoxia. Then, the intracellular level of αKG was tested by αKG determination kit (C) (n = 3). Cells were collected and indicated antibodies were applied to test HIF1α, EGLN1 and GDH1 proteins (D).
  • E
    HCT116 and SW480 cells with or without GDH1 were supplemented with cell permeable methyl‐αKG under normoxia and hypoxia. Then, cell viability was counted by trypan blue staining (n = 3).
  • F, G
    HCT116 and SW480 cells with or without GDH1 were cultured under normoxia or hypoxia. Cells were collected for determining glutamate (Glu, F) and ammonia (NH4Cl, G) (n = 3).
  • H
    HCT116 were incubated with or without cell permeable octyl‐2HG under normoxia or hypoxia. Indicated antibodies were applied to test HIF1α, EGLN1 and GDH1 proteins (n = 3).
  • I
    HIF1α and GDH1 were tested by western blot in HCT116 or SW480 cells with Vec or GDH1 overexpression under normoxia and hypoxia.
  • J
    HCT116 and SW480 cells with or without GDH1 overexpression were incubated under normoxia and hypoxia, respectively. Then, cell viability was counted by trypan blue staining (n = 3).
  • K
    The apoptotic percent of cells in Fig S1D was counted by PI/Annexing V double staining (n = 3).
  • L
    TUNEL analysis of GDH1 knockdown HCT116 cells under hypoxia. The left was the representative microscopic data, the right was the calculated relative intensity data (n = 3).
  • M
    The overall αKG level in HCT116 or SW480 cells with or without GDH1 overexpression under normoxia and hypoxia (n = 3).

Data information: Data are mean ± SD from the biological replicates (B, C, E–H, J–M). Statistics: two‐way ANOVA with Tukey's HSD post hoc test (B, E, J); one‐way ANOVA with Tukey's HSD post hoc test (C, F–H, M); unpaired two‐tailed Student's t‐test (K, L).

Source data are available online for this figure.