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. 2016 Oct 6;291(46):24105–24120. doi: 10.1074/jbc.M116.751263

FIGURE 6.

FIGURE 6.

Forced GFAT1 expression potentiates HIF-1α expression and lactate production. A, Western blot analysis for the expression of GFAT1, HIF-1α, and LDHA in GFAT1 transfectants. Has2+Neo cells were transfected with expression plasmids carrying human GFAT1 to establish stable transfectants expressing various levels of GFAT1. Cell lysates from the GFAT1 transfectants were subjected to Western blotting analysis. β-Actin was used as an internal control. B, production of UDP-HexNAc by forced GFAT1 expression. GFAT #1 (negative) and #10 (high) cells were cultured for 24 h in glucose-free DMEM and then further incubated in normal culture medium for up to 6 h. UDP-HexNAc production was monitored using HPLC and compared between GFAT #1 (open bars) and #10 (black bars) cells. Data represent the mean ± S.D. (error bars) of three independent experiments. *, p < 0.05. C, LDH activity in the GFAT1 transfectants. Cell lysates from the GFAT1 transfectants were measured for LDH activity. Data represent the mean ± S.D. of three independent experiments. *, p < 0.05; **, p < 0.01 as compared with the GFAT #1 clone. D, lactate production in the GFAT1 transfectants. Conditioned media from the GFAT1 transfectants were measured for lactate production. Data represent the mean ± S.D. of three independent experiments. **, p < 0.01 as compared with the GFAT #1 clone.