Journal: Proteomes

Article Title: Calcium Homeostasis and Muscle Energy Metabolism Are Modified in HspB1-Null Mice

doi: 10.3390/proteomes4020017

Figure Lengend Snippet: Suppliers and conditions for each antibody used in this study.

Article Snippet: HspA9 , Stress-70 protein, mitochondrial , Monoclonal , R & D Systems: MAB3584 , 1/200.

Techniques:

Journal: Signal Transduction and Targeted Therapy

Article Title: GRP75 triggers white adipose tissue browning to promote cancer-associated cachexia

doi: 10.1038/s41392-024-01950-w

Figure Lengend Snippet: 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 )

Article Snippet: Mouse IL-6 (abs520004) and TNF-α (abs520010) ELISA kits were purchased from Absin Biotechnology Co., Ltd. (Shanghai, China); mouse GRP75 ELISA kits (NBP2-76446) were obtained from Novus Biologicals (USA); and human GRP75 ELISA kits (ELH-HSPA9-A) were obtained from Raybiotech, Inc. (USA).

Techniques: Staining, Expressing, Western Blot, Derivative Assay, Negative Control, Protein Concentration, Quantitation Assay, In Vivo, Injection, Over Expression, Control, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: GRP75 triggers white adipose tissue browning to promote cancer-associated cachexia

doi: 10.1038/s41392-024-01950-w

Figure Lengend Snippet: GRP75 binds to and stabilizes ANT2 by decreasing its ubiquitination level. a Identification of endogenous GRP75-interacting proteins. Extracts from the iWAT of YES2 tumour-bearing mice were incubated with protein A/G magnetic beads conjugated with an anti-GRP75 antibody. The eluted proteins were resolved by SDS‒PAGE and visualized by silver staining. The differential gel piece framed by the red rectangle was analysed by mass spectrometry. b Coimmunoprecipitation of endogenous ANT2 and GRP75 in the iWAT of YES2 tumour-bearing mice. Anti-IgG was used as a negative control. c Coimmunoprecipitation of GRP75 and ANT2 in differentiated 3T3-L1 adipocytes transfected with Myc-GRP75 plasmids. d PLA signals and quantification of the combination of anti-GRP75 and anti-ANT2 in differentiated 3T3-L1 adipocytes treated with NC-EVs or GRP75-overexpressing EVs. DAPI, nuclei. Scale bar, 10 μm. e Immunoblotting with anti-GST antibodies was used to detect the binding of recombinant His-ANT2 to GST-GRP75. f Coimmunoprecipitation of ANT2 and truncated domains of GRP75 (bottom panel). Flag-tagged wild-type GRP75 (GRP75-FL) or truncated GRP75 (GRP75-N and GRP75-C) were transfected into HEK293T cells. g Immunoblots (left panel) and real-time qPCR analysis (right panel) of ANT2 and GRP75 in differentiated 3T3-L1 adipocytes transfected with control vector (NC) or Myc-GRP75 plasmids. h Evaluation of the ANT2 half-life in GRP75-overexpressing adipocytes treated with cycloheximide. The indicated proteins were analysed by immunoblotting, and the quantification of ANT2 protein relative to the control β-actin by ImageJ software is shown at the bottom. i Ubiquitination assays of exogenous ANT2 in lysates from differentiated 3T3-L1 adipocytes transfected with NC or GRP75 plasmids. The data are presented as the mean ± SEM. The exact P values were tested with an unpaired two-tailed Student’s t test in ( g )

Article Snippet: Mouse IL-6 (abs520004) and TNF-α (abs520010) ELISA kits were purchased from Absin Biotechnology Co., Ltd. (Shanghai, China); mouse GRP75 ELISA kits (NBP2-76446) were obtained from Novus Biologicals (USA); and human GRP75 ELISA kits (ELH-HSPA9-A) were obtained from Raybiotech, Inc. (USA).

Techniques: Incubation, Magnetic Beads, Silver Staining, Mass Spectrometry, Negative Control, Transfection, Western Blot, Binding Assay, Recombinant, Control, Plasmid Preparation, Software, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: GRP75 triggers white adipose tissue browning to promote cancer-associated cachexia

doi: 10.1038/s41392-024-01950-w

Figure Lengend Snippet: 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 )

Article Snippet: Mouse IL-6 (abs520004) and TNF-α (abs520010) ELISA kits were purchased from Absin Biotechnology Co., Ltd. (Shanghai, China); mouse GRP75 ELISA kits (NBP2-76446) were obtained from Novus Biologicals (USA); and human GRP75 ELISA kits (ELH-HSPA9-A) were obtained from Raybiotech, Inc. (USA).

Techniques: Quantitation Assay, Transfection, Control, Plasmid Preparation, Western Blot, Expressing, Proximity Ligation Assay, Immunoprecipitation, FLAG-tag, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: GRP75 triggers white adipose tissue browning to promote cancer-associated cachexia

doi: 10.1038/s41392-024-01950-w

Figure Lengend Snippet: 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 )

Article Snippet: Mouse IL-6 (abs520004) and TNF-α (abs520010) ELISA kits were purchased from Absin Biotechnology Co., Ltd. (Shanghai, China); mouse GRP75 ELISA kits (NBP2-76446) were obtained from Novus Biologicals (USA); and human GRP75 ELISA kits (ELH-HSPA9-A) were obtained from Raybiotech, Inc. (USA).

Techniques: In Vitro, In Vivo, Quantitation Assay, Protein Concentration, Western Blot, Transfection, Injection