Fig. 3
From: GRP75 triggers white adipose tissue browning to promote cancer-associated cachexia
GRP75 is essential for adipocyte browning. a Five most representative subcellular localization analyses of 63 overlapping proteins. b Representative IHC images and the staining scores of GRP75 (top) in 63 pairs of patient tumours (T) and matched adjacent normal tissues (N). Scale bar, 200 μm. c Kaplan‒Meier survival analysis of patients with ESCC stratified by GRP75 expression (n = 107; P < 0.001, log-rank test). d Immunoblots of GRP75 in EVs derived from mouse colon cancer cells (MC38 and C26), ESCC cells (YES2 and KYSE150) and other cachexia-inducing tumour cells (HepG2, LLC, AsPC-1, and BxPC3). e MtDNA copy number was detected in differentiated 3T3-L1 adipocytes treated with NC-EVs or GRP75-EVs. f Oxygen consumption rate (OCR) in primary adipocytes treated by GRP75-EVs or GRP75-EVs with si-UCP1. Primary adipocytes treated with NC-EVs are shown as a negative control. Left: plot of the time course OCR normalized to the protein concentration. Right: calculated respiration levels of basal and proton leaked respiration. g Quantitation of intracellular TG contents in adipocytes treated as described in f. h Immunoblots of GRP75 and UCP1 in differentiated 3T3-L1 adipocytes treated as described in f. i Schematic diagram of in vivo subcutaneous injection of PBS, KYSE150 cells with stable GRP75 overexpression (LV-Flag-GRP75) or negative lentiviral vectors (LV-Control) in six-week-old BALB/c-nude mice (n = 6 per group). j NBWs of the PBS, LV-Control and LV-Flag-GRP75 groups. k Ratios of iWAT, eWAT, and iBAT to NBW in the three groups. l Representative H&E-stained images of iWAT from the three groups. Scale bar, 50 μm. Quantified adipocyte sizes are shown on the right side. m, n Oxygen consumption volume and heat production of the LV-Control and LV-Flag-GRP75 groups in the metabolic cage housed at 22°C (n = 6 per group). o Rectal temperatures of the PBS, LV-Control and LV-Flag-GRP75 groups. p Representative IHC images of UCP1 and GRP75 staining in iWAT from the three groups. Scale bar, 50 μm. The magnified image labelled with a red rectangle is shown on the upper right side. Scale bar, 25 μm. q Immunoblotting of GRP75 and UCP1 protein in iWAT from three groups (n = 4; representative of four biological replicates per group). The data are presented as the mean ± SEM. The exact P values were tested with one-way ANOVA in (j–l, o) and unpaired two-tailed Student’s t test in (m and n)
