Extended Data Fig. 2: Localization of the mmBCFA synthetic proteins FASN and CRAT to peroxisomes.
From: Peroxisomal metabolism of branched fatty acids regulates energy homeostasis
a, Schematic of mmBCFA biosynthesis using short branched acylCoA (BrCoA) derived from mitochondrial catabolism of BCAA. b, Western blot analysis of CRISPR/Cas9-mediated KO of BCKDHα in brown adipocytes. N = 2. c-d, Mass spectrometry analysis of mmBCFA (c) and conventional fatty acids (d) in control and BCKDHA KO brown adipocytes. N = 4. e, mRNA levels of mmBCFA β-oxidation and synthesis genes in iWAT of WT mice maintained at thermoneutrality or 4 °C for 7 days. N = 4. f, FASN gene expression in control or norepinephrine (NE)-treated differentiated mouse UCP1−/− brown adipocytes. N = 3. g, FASN gene expression in cultured human brown-like adipocytes treated with or without forskolin. N = 3. h, Immunofluorescence analysis of peroxisomal localization of FASN in wild-type brown adipocytes after NE treatment for 0 or 2 h. N = 3. Scale bar, 10 mm. i, Immunofluorescence analysis of peroxisomal localization of FASN in BAT of WT mice treated with or without CL316,243. N = 3. Scale bar, 10 mm. j, Co-immunoprecipitation of HA-PEX7 with FLAG-FASN in HEK293T cells. k, Immunofluorescence analysis of peroxisomal localization of CRAT in wild-type brown adipocytes after NE treatment for 2 h. Scale bar, 10 mm. l, Western blot analysis of CRISPR/Cas9-mediated KO of CRAT in brown adipocytes. N = 3. m, Oil Red O staining in differentiated CRAT KO and control adipocytes. Scale bar, 300 mm. n,o, Mass spectrometry analysis of mmBCFA (n) and stCFA (o) in control and CRAT KO brown adipocytes. N = 3. p, OCR measurement in sgCRAT and control brown adipocytes at baseline and after sequential treatment with leucine, NE, and iso-C17:0. N = 8 (sgControl); N = 7 (sgCRAT). q, Gene expression analysis in sgCRAT and control brown adipocytes overexpressing ACOX2 or GFP. N = 3. r, OCR measurement in sgCRAT and control brown adipocytes overexpressing ACOX2 or GFP. Oligo, oligomycin; FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; AA + R, antimycin A and rotenone. N = 9. Data with error bars are mean ± SEM. Data in b–i, k, l, and n–r are from biologically independent samples. Statistical significance was determined by two-sided unpaired Student’s t test (c–i, k, n–o), two-way ANOVA with Sidak’s (p) or Tukey’s test (r), and one-way ANOVA with Fisher’s LSD (q). Representative images in h–k, and m are from two independent experiments. Panel a was created using BioRender (https://biorender.com).
