Extended Data Fig. 1: PFKL is required for the interaction between PLIN2 and CPT1A, lipid droplet-mitochondria tethering, and lipid droplet lipolysis.
b, c, e, g, j, Immunoblotting analyses with the indicated antibodies were performed, presenting representative results from three independent experiments. d, i, k, l, Representative images from three independent experiments. a, LDs from Huh7 cells were purified after 40 min of glucose deprivation and subjected to LC-MS/MS. Selected peptide hits of LD-associated proteins are shown. b, LM3 cells were treated with 40 min of glucose deprivation. c, Huh7 cells with depleted endogenous PLIN2 and reconstituted expression of Flag-rPLIN2 protein were treated with glucose deprivation for the indicated period of time. d, Representative TEM images of Huh7 cells treated with 1 h of glucose deprivation were presented (left). Percentages of mitochondria-associated LDs per field were quantified. n = 15 or 16 microscope fields (right). e, Huh7 cells were infected with lentiviruses expressing shCtrl or shCPT1A. f, Huh7 cells with glucose deprivation for the indicated period of time were subjected to glycolytic metabolites analysis by LC-MS/MS. n = 3. g, Huh7 cells expressing Flag-PLIN2 were treated with 40 min of glucose deprivation. Cell lysates were incubated with glycolytic metabolites (F6P, FBP, DHAP, G3P, 3PG) followed by immunoprecipitation. h, PFKL expression levels were analyzed in normal liver and HCC tissues using a TCGA dataset. Normal: minimum, 22.235; maximum, 83.356; lower quartile, 38.085; upper quartile, 61.781; and median 48.352; HCC: minimum, 13.449; maximum, 166.926; lower quartile, 52.154; upper quartile, 98.358; and median, 72.891. The horizontal lines mark the median. n (Normal) = 50, n (HCC) = 371. i, Workflow for purifying LDs from Huh7 cells expressing GFP-Flag-LiveDrop (left). Immunofluorescence analyses were performed (right). j, LM3 cells stably expressing shCtrl or shPFKL were transfected with Flag-PLIN2 and were treated with 40 min of glucose deprivation (left). PFKL depletion efficiency was quantified. n = 3 (right). k, LM3 cells stably expressing shCtrl or shPFKL#1 were stimulated by 40 min of glucose deprivation (left). Percentages of mitochondria-associated LDs per cell were quantified. n (shCtrl) = 20, n (shPFKL) = 20 (right). l, m, LM3 cells stably expressing shCtrl or shPFKL#1 were treated with 4 h glucose deprivation for (l). LDs number and the percentage of LDs area per cell were quantified. n (shCtrl, Glc+) = 30, n (shCtrl, Glc‒) = 30, n (shPFKL, Glc‒) = 30 (m). d, f, h, j, k, m, Data are presented as means ± SD of three biologically independent replicates, analyzed by two-sided unpaired Student’s t-test (h, j, k) or one-way ANOVA (m).
