Fig. 6
From: GRP75 triggers white adipose tissue browning to promote cancer-associated cachexia
GRP75 inhibitors alleviate adipocyte browning in vitro and in vivo. a Quantitation of intracellular TG content in differentiated 3T3-L1 adipocytes treated with NC-EVs or GRP75-EVs in the presence or absence of the GRP75 inhibitor withanone (WNN). b OCR of differentiated adipocytes as described in a. Left: plot of the time course OCR normalized to the protein concentration. Right: calculated respiration levels of basal and proton-leaked OCRs. c Immunoblots of GRP75, ANT2, and UCP1 in differentiated adipocytes, as shown in a. d Coimmunoprecipitation of GRP75 and ANT2 in HEK293T cells treated with or without WNN. HEK293T cells were transfected with GFP-GRP75 plasmids and Flag-ANT2 plasmids. e Experimental design of the in vivo experiment. YES2 tumour-bearing mice were intraperitoneally injected with DMSO (DMSO), WNN (5 mg/kg), cisplatin (10 mg/kg, CDDP), or a combination of WNN with cisplatin (Combo). The PBS and PF groups included tumour-free mice injected with vehicle. f NBWs of the six groups described in e on day 48. g Ratios of iWAT (left) and eWAT (right) to NBW in e on day 48. h Representative IHC images of UCP1 in the iWAT from six groups. Scale bar, 50 μm. Magnified images labelled with a red rectangle are shown in the bottom panel. Scale bar, 25 μm. i–j Immunoblots of GRP75, ANT2, and UCP1 in the iWAT of the DMSO and WNN groups (i) or the CDDP and Combo groups (j) (n = 6; representative of six biological replicates per group). k Working model for how tumour extracellular vesicular GRP75 regulates white adipocyte browning in cachexia progression (created by Biorender.com). The data are presented as the mean ± SEM. n = 6 (PBS and PF), n = 8 (DMSO), n = 12 (WNN), n = 10 (CDDP) and n = 9 (Combo). The exact P values were tested with one-way ANOVA in (a, b, f, and g)
