Fig 2: HPD promotes G6PD gene expression by controlling AMPK activity.a Cell lysates from HPD-knockdown H1299 cells were tested for phosphorylation levels of AMPK (pT172) and ACC1 (p79) by western blotting. b HEK293 cells were cotransfected with Flag-HDAC10 with or without HA-AMPK. HDAC10 was pulled down with Flag beads and coprecipitated proteins were analyzed by immunoblotting with anti-Flag or anti-HA antibody. The expression levels of transfected constructs were analyzed by immunoblotting. c Immunoprecipitation was conducted in AMPK (−/−) and AMPK (+/+) cell line MEF. In all, 2 mg of cell lysate was immunoprecipitated with anti-HDAC10 and immunoblotted using anti-HDAC10 or anti-phospho-(Ser/Thr). Sixty micrograms of cell lysate was used as an input control. d H1299 cells were cotransfected with Flag-HDAC10 WT with HA-AMPK in the presence of Flag-HPD. Flag-HDAC10 was pulled down with Flag beads and co-immunoprecipitated proteins were analyzed by immunoblotting with anti-Flag or anti-phospho-(Ser/Thr) antibody. The expression levels of transfected constructs were analyzed by immunoblotting. e H1299 cells were cotransfected with Flag-HDAC10 double S393/540 mutants (SA) with HA-AMPK in the presence of Flag-HPD. HDAC10 was pulled down with Flag beads and coprecipitated proteins were analyzed by immunoblotting with anti-Flag or anti-phospho-(Ser/Thr) antibody. The expression levels of transfected constructs were analyzed by immunoblotting. f G6PD protein levels were analyzed by western blotting in HPD stable-expressing H1299 cells and control vector cells with Compound C treatment. g G6PD protein levels were analyzed by western blotting in HPD stable-expressing H1299 cells infected with lentivirus harboring AMPK shRNA. h G6PD protein levels were analyzed by western blotting in HPD-knockdown H1299 cells and control vector cells with AICAR treatment. i Cell lysates from HPD-knockdown H1299 cells were tested for phosphorylation levels of AMPK (pT172), ACC1 (p79), and G6PD by western blotting

Fig 3: HPD reprograms oxidative PPP.a–f HPD knockdown and control cells harboring an empty vector were tested for oxidative PPP flux (a), RNA biosynthesis (b) and NADPH/NADP+ ratio (c), glycolysis ratio (d), lactate production (e), as well as ROS level (f). The error bars represent mean values ± SD from three independent experiments (*0.01 < p < 0.05; **0.001 < p < 0.01; ***p < 0.001)

Fig 4: HPD promotes G6PD gene expression by controlling HDAC10 phosphorylation.a The purity of the cytosolic and nuclear fractions from knockdown and control cells harboring an empty vector was tested for HDAC10 localization. b Immunoprecipitation of HDAC10 and western blot to detect phosphorylation levels of HDAC10 in HPD stable-expressing H1299 cells and control vector cells. c G6PD protein levels were analyzed by western blotting in H1299 cells, which were transiently transfected with vectors encoding HDAC10 WT, S373, S393, and S540 mutants. d G6PD protein levels were analyzed by western blotting in H1299 cells, which were transiently transfected with vectors encoding HDAC10wt, S368, and double S393/540 mutants (SA). e G6PD protein and H3 acetyaltion levels were analyzed by western blotting in HPD stable-expressing H1299 cells that were transiently transfected with vectors encoding HDAC10 double S393/540 mutants (SA). f Purity of the cytosolic and nuclear fractions from H1299 cells, which were transiently transfected with vectors encoding HDAC10 WT and HDAC10 double S393/540 (SA) mutant

Fig 5: HPD contributes to cell proliferation through upregulation of G6PD.a Determination of G6PD mRNA expression levels by qRT-PCR (left), protein levels by western blotting (right upper), and mRNA expression levels by RT-PCR (right lower) in HPD-knockdown and control H1299 cells. b Determination of G6PD mRNA expression levels by qRT-PCR (left), protein levels by western blotting (right upper), and mRNA expression levels by RT-PCR (right lower) in HPD-knockdown and control H226 cells. c–e G6PD knockdown and control cells harboring an empty vector were tested for oxidative PPP flux (c), RNA biosynthesis (d), and NADPH/NADP+ ratio (e). f–g Overexpression of G6PD in HPD-knockdown cells restores decreased oxidative PPP flux (f) and RNA biosynthesis (g). h–i Cell proliferation rates determined by cell counting in human lung cancer H1299 (h) and H226 (i) cells with stable-knockdown HPD and overexpression of G6PD. The error bars represent mean values ± SD from three independent experiments (*0.01 < p < 0.05; **0.001 < p < 0.01; ***p < 0.001)