Extended Data Fig. 4: PFKL-mediated PLIN2 S159 phosphorylation promotes the interaction between PLIN2 and CPT1A and lipid droplet-mitochondria tethering.
a, c, Immunoblotting analyses with the indicated antibodies were performed, presenting representative results from three independent experiments. d, f, g, Representative images from three independent experiments. a, LM3 cells with depleted endogenous PLIN2 and reconstituted expression of Flag-rPLIN2 proteins were treated with 40 min of glucose deprivation. b, A schematic depicting Red C12 transport assay. c, d, e, Flag-rPLIN2proteins were expressed in Huh7 cells with depletion of endogenous PLIN2. Immunoprecipitation analyses with an anti-Flag antibody were performed (c). Cells were labeled with GFP-LiveDrop and mito-DsRed. Percentages of mitochondria-associated LDs per cell were quantified. n (WT) = 32, n (S159D) = 36 (d). LDs number and the percentage of LDs area per cell were quantified. n (WT) = 55, n (S159D) = 56 (e). f, Huh7 cells with depleted endogenous PFKL and reconstituted expression of Flag-rPFKL proteins were stimulated by 40 min of glucose deprivation (left). Percentages of mitochondria-associated LDs per cell were quantified. n (WT) = 26, n (T331A) = 28. g-i, Huh7 cells with depleted endogenous PFKL and reconstituted expression of Flag-rPFKL proteins were incubated with Red C12 for 16 h before treatment with 4 h of glucose deprivation (g). Mitochondrial translocation of Red-C12 from LDs upon glucose starvation was quantified. n (WT) = 30, n (T331A) = 30. A.U., arbitrary unit (h). LDs number and the percentage of LDs area per cell were quantified. n (Glc+) = 30, n (Glc‒) = 30 (i). d-f, h, i, Data are presented as means ± SD of three biologically independent replicates, analyzed by two-sided unpaired Student’s t-test (d-f, h) or two-way ANOVA (i).
