Extended Data Fig. 3: PHGDH negatively regulates nuclear NAD+ upon glucose deprivation.
From: The alternative activity of nuclear PHGDH contributes to tumour growth under nutrient stress
In g-i, immunoblotting analyses were performed using the indicated antibodies. In a-f, data are presented as mean ± s.d. (n = 3 independent experiments), statistical analysis was performed using the two-tailed Student’s t-test. (a) SW1990 cells with or without depletion of PHGDH were transfected with nucleus-targeting sequence containing mCherry-FiNad (Nuc-mCherry-FiNad) and mCherry-cpYFP (Nuc-mCherry-cpYFP) (left panel) or SoNar (Nuc-SoNar) and iNapc (Nuc-iNapc) (right panel). Cells were cultured in the normal or glucose-free DMEM for 12 h. Normalized ratio of fluorescence intensities excited at 485 nm and 590 nm (F485nm/F590nm indicates NAD+) and fluorescence intensities excited at 420 nm and 485 nm (F420nm/F485nm indicates NADH/NAD+) were recorded by Microplate Reader. (b) SW1990 (left panel) or LM3 (right panel) cells with depletion of PHGDH and reconstituted with indicated Flag-tagged rPHGDH were cultured in the normal or glucose-free DMEM for 36 h. Cellular viability was examined by CCK-8 assay. (c, d) SW1990 cells with or without depletion of PHGDH (c) or SW1990 cells with depletion of PHGDH and reconstituted with indicated Flag-tagged rPHGDH (d) were cultured in the normal or glucose-free DMEM for 12 h. The NAD+ (left panels) and NADH/NAD+ (right panels) content was measured using the NAD+ assay kit. (e, f) SW1990 cells with or without PHGDH depletion (e) or with depletion of PHGDH and reconstituted expression of indicated Flag–rPHGDH (f) were transfected with mCherry-FiNad and mCherry-cpYFP (left panels) or SoNar and iNapc (right panels). Cells were cultured in the normal or glucose-free DMEM for 12 h. Normalized ratio of fluorescence intensities was recorded by Microplate Reader. (g) SW1990 (left panel) or LM3 (right panel) cells with or without depletion of PHGDH were cultured in the normal or glucose-free DMEM for 12 h. (h) LM3 cells with depletion of PHGDH and reconstituted with indicated Flag-tagged rPHGDH were cultured in the normal or glucose-free DMEM for 12 h. (i) SW1990 cells with or without depletion of PHGDH were transfected with or without si-NMANT1. Cells were cultured in the glucose-free DMEM for 12 h.
