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. 2023 Sep 29;14:6099. doi: 10.1038/s41467-023-41849-8

Fig. 7. TMEM135 regulates mitochondrial fission in human thermogenic adipocytes.

Fig. 7

a qPCR analysis of Tmem135 expression in human brown adipocytes (hBAT) treated with vehicle (n = 3) or 10 µM forskolin (n = 3) for 4 h. b Western blot analysis of TMEM135 knockout using CRISPR/Cas9 in human brown adipocytes; n = 3. c Analysis of mitochondrial morphology using confocal microscopy in control (n = 5) and TMEM135 KO (n = 6) human brown adipocytes stained with MitoTracker Green. Plot shows quantification of mitochondrial morphology. Scale bar: 10 μm. d mtDNA copy number normalized to nuclear DNA in control (n = 6) and TMEM135 KO (n = 6) human brown adipocytes. e OCR measured in control (n = 10) and TMEM135 KO (n = 10) human brown adipocytes using a Seahorse XF24 Extracellular Flux Analyzer; oligo oligomycin, FCCP carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, AA + R mixture of antimycin A and rotenone. f Relative gene expression of TMEM135 in abdominal subcutaneous WAT of female lean (n = 9), obese/insulin sensitive (IS) (n = 21), and obese/insulin resistance (IR) (n = 18) patients, based on analysis of publicly available transcriptome-profiling data (GEO: GSE94753). g A coding variant of TMEM135 associated with increased BMI in a Hispanic population identified in a publicly available AMP-T2D-GENES exome sequencing dataset. h Domain architecture and sequence alignment of human, bovine and mouse TMEM135. i Western blot analysis of TMEM135 KO human adipocytes transduced with lentivirus expressing GFP (n = 3), mouse WT (n = 3) or G369E TMEM135 (n = 3). j Confocal microscopy analysis of mitochondrial morphology of TMEM135 KO human adipocytes transduced with lentivirus expressing GFP (n = 5), mouse WT (n = 4) or G369E TMEM135 (n = 4). Plot shows quantification of mitochondrial morphology. Scale bar: 5 μm. Data are presented as mean ± SEM; statistical significance was determined by two-tailed unpaired Student’s t test (a, c, d, f, j) or 2-way ANOVA with Bonferroni’s post hoc test (e).