Fig 2: PFKP promotes mitochondrial recruitment of AMPK and enhances the phosphorylation of ACC2.a PFKP IP in H1299 cells under control or GS. b Immunofluorescence analysis of mito-GFP and PLA puncta (interacted PFKP and AMPK) in H1299 cells under control (Ctrl) or GS for 4 h. The gray values of green and red signals between two the arrows were plotted. Scale bar, 10 µm. c, d Mitochondria fraction analysis of H1703 (c) and H1299 (d) cells under control or GS. e IP of ACC1 and ACC2 in H1703 cells under control or GS. f ACC2 IP in H1299 cells under control or treated with GS for 4 h or 8 h. Data are shown as means ± SD with three biological replicates. ns, no significance; *P < 0.05, **P < 0.01, ***P < 0.001 by two-tailed Student’s t-test.

Fig 3: PFKP promotes NSCLC cell survival under GS via long-chain fatty acid oxidation.a Propidium iodide (PI) exclusion test of H1299 cells under control (Ctrl) or GS for 48 h. FSC: forward scatter. b Cell viability of H1299 (n = 9) or H1703 cells (n = 8) under control (Ctrl) or GS for 48 h. c Cell viability of H1299 with PFKP knock-down (KD) (n = 5) under control (Ctrl) or GS for 48 h. d Cell viability of H1299 KO cells with ACC2 KD (n = 9) under control (Ctrl) or GS for 48 h. e Cell viability of H1703 cells with AMPKa KD (n = 4) under control (Ctrl) or GS for 8 h. f Cell viability of H1703 cells treated with 1 mM PFKP inhibitor 2,5-Anhydro-D-glucitol-1,6-diphosphate (n = 4) under control (Ctrl) or GS for 24 h. g Cell viability of H1703 PFKP KO cells transfected with empty vector, Domain 1, or Flag-HA tagged PFKP (n = 4) under control (Ctrl) or GS for 24 h. h Cell viability of H1703 treated with 5 mM N-acetylcysteine (NAC) (n = 4) under control (Ctrl) or GS for 24 h. Data are shown as means ± SD with n indicating the number of biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001 by two-tailed Student’s t-test.

Fig 4: PFKP promotes long-chain fatty acid oxidation via ACC2 to maintain energy and redox homeostasis.a Relative level of long-chain free fatty acids (LCFFAs) in H1299 (n = 4) or H1703 (n = 4) cells under GS for 14 h over that in control. b Relative concentration of lauric acid (C12:0), myristic acid (C14:0), palmitic acid (C16:0), stearic acid (C18:0), arachidic acid (C20:0), and behenic acid (C22:0) in H1299 (n = 4) or H1703 (n = 5) cells under GS for 14 h over that in control. c–e Relative ATP (c, n = 4), NADPH (d, n = 4), and ROS level (e, n = 6) in H1299 or H1703 cells under GS for 14 h over that in control (Ctrl). Data are shown as means ± SD with n indicating the number of biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001 by two-tailed Student’s t-test.