Fig. 5
From: GRP75 triggers white adipose tissue browning to promote cancer-associated cachexia
ANT2–GRP75 promotes adipocyte browning by enhancing the interaction with UCP1 and its stability. a, b Quantitation of intracellular TG (a) and ATP (b) levels in primary adipocytes transfected with control vector (NC) or Flag-ANT2 plasmids (pANT2). c Immunoblots showing ANT2 and UCP1 expression in differentiated 3T3-L1 adipocytes treated as described in a. d, e Quantitation of intracellular TG (d) and ATP (e) levels in primary adipocytes treated with NC-EVs or GRP75-EVs and transfected with si-NC or si-ANT2. f Immunoblots showing GRP75, ANT2, and UCP1 expression in differentiated 3T3-L1 adipocytes treated as described in d. g Proximity ligation assay signals and enlarged images of the combination of anti-ANT2 and anti-UCP1 in differentiated 3T3-L1 adipocytes treated with NC-EVs or GRP75-overexpressing EVs. DAPI, nuclei. Scale bar, 10 μm. h Immunoblots of UCP1 in differentiated 3T3-L1 adipocytes transfected with NC or Flag-ANT2 plasmids in the presence of cycloheximide. i Ubiquitination assays of UCP1 in lysates from differentiated 3T3-L1 adipocytes transfected with NC or Flag-ANT2 plasmids. j Coimmunoprecipitation of GRP75, ANT2, and UCP1. HEK293T cells were co-transfected with Myc-GRP75, ANT2, and Flag-UCP1. Protein extracts were immunoprecipitated with antibodies against Myc-tag or Flag-tag, followed by immunoblotting with the indicated antibodies. k Exogenous GRP75 enhances the ANT2–UCP1 interaction. Coimmunoprecipitation assays were performed against ANT2 using HEK293T cell lysates with or without transfection of Myc-GRP75. The quantification of protein relative to the control protein β-actin by ImageJ are shown at the bottom in (c, f and h). The data are presented as the mean ± SEM. The exact P values were tested with unpaired two-tailed Student’s t tests in (a and b) and one-way ANOVA in (d and e)
