Fig 1: STX4 can localize to the inner mitochondria membrane and regulates UCP1 protein stability.a Representative immunoblotting of Tubulin, GM130, TOM70, STX4 and Caveolin in cytosol fraction, mitochondria fraction and non-mitochondrial membrane fraction from WT and STX4KO brown adipocytes at day 11 of differentiation. b Representative immunoblotting of TOM70, STX4, COX IV and Caveolin in mitochondria fraction from male WT (2-month-old) brown adipose tissue treated with 20 µg/µl proteinase K for 0 min, 10 min, 20 min and 30 min. c Representative immunoblotting of TOM70, STX4 and UCP1 in mitochondria fraction from male WT and STX4KO brown adipocytes at day 12. d The fold change of the log2 ratio of the heavy (H) isotope labeled UCP1 protein divided by the unlabeled light protein (L) at day 12 compared to day 11 in WT and STX4KO adipocytes. n = 3 biologically independent samples. p = 0.023. e Lysates from 293 T cell overexpressing STX4-Myc and UCP1-Flag for 24hrs were subjected to immunoprecipitation using ether Flag antibody or Myc antibody, followed by blotting with antibodies against Myc, UCP1, SNAP23, ATP5A and TOM70. f The 293 T cell overexpressed STX4-Myc and UCP1-Flag for 24 h were immunostaining for Flag (green) and Myc (red) with DAPI (blue) labeling of nuclei. These pictures are representative of 50 cells. Scale bar: 20 µm and 5 µm (insert). g The extent of co-localized STX4-Myc and UCP1-Flag was determined by Pearson’s coefficient from 3 independent experiments with the quantification from 50 cells/experiment. h Hypothetical schematic model that accounts for the loss of STX4 mediating both mitochondrial and glycolytic dysfunction necessary for the activation of brown adipocyte pyroptosis (Created with BioRender.com). Each experiment has been repeated for 3 times. All data represent the mean ± SEM. *p < 0.05, ** p < 0.01, and ***p < 0.001, by two-tailed Student’s t test.