Fig. 2. TMEM135 is the most decreased protein in mitochondria of peroxisome-deficient BAT and its mitochondrial localization is mediated by peroxisome-derived lipids.

a Schematic diagram of proteomic analysis in mitochondria isolated from BAT of cold-treated Pex16-AKO and control mice. Created with BioRender.com. b, c Heat map and volcano plot analysis of mitochondrial proteomics in BAT of Pex16-AKO and control mice; n = 5/group. d, e qPCR and Western blot analysis of TMEM135 in BAT of control and Pex16-AKO mice after cold exposure; control: n = 9; Pex16-AKO: n = 10. f Immunofluorescence analysis in control and Pex16-AKO brown adipocytes expressing mito-GFP stained with an antibody against TMEM135. Colocalization was quantified using ImageJ. Quantification was performed with the investigator blinded to the identity of the samples. Scale bar: 10 μm. The images are representative of four independent experiments and the quantification is based on a total of 54 control and 64 Pex16-AKO cells. g qPCR analysis of Gnpat and Tmem135 in differentiated BAT SVF cells treated with lentivirus-encoding scrambled (SC) shRNA (n = 3), GNPAT shRNA (n = 3) or GNPAT shRNA plus AG (n = 3). h Immunofluorescence analysis using an antibody against TMEM135 in the brown adipocytes expressing Mito-GFP treated with lentivirus-encoding scrambled (SC), GNPAT shRNA or GNPAT shRNA plus AG. Colocalization was quantified using ImageJ. Scale bar: 10 μm. The images are representative of three independent experiments and the quantification is based on a total of 10 cells per group. Data are presented as mean ± SEM; statistical significance was determined by two-tailed unpaired Student’s t test (d, f–h). Comparisons between groups were made with a two-tailed unpaired Student’s t-test adjusted for multiple comparisons using the Benjamini-Hochberg method (c).