anti human β2 antibody  (ATCC)

Journal: Nature communications

Article Title: The autophagy component LC3 regulates lymphocyte adhesion via LFA1 transport in response to outside-in signaling.

doi: 10.1038/s41467-025-56631-1

Figure Lengend Snippet: Fig. 1 | Identification of factors colocalizing with LFA1 intracellular clusters. a LFA1 (αLβ2) intracellular cluster (ICC) formation concomitant with lymphocyte adhesion. Adhesion of lymphocytes (BAF/LFA1) to the ICAM1-displaying surface via LFA1 results in the formation of LFA1 ICCs inside the cell. LFA1 was visualized by fusing a SNAP tag to the C-terminus of β222. b Colocalization of β2-SNAP (shown in magenta) with factors involved in the regulation of vesicular trafficking (shown in green). Colocalized spots are indicated by arrows. Colocalization of LFA1 (β2) with TGN38 (TGN, n = 15), Rab7 (n = 9) or LC3 (n = 12) was quantified using Mander’s coefficient22. c Co-trafficking of β2-SNAP and CLIP-LC3 in adhered lymphocytes. Yellow arrows and cyan arrowheads indicate the ICCs containing both β2 and LC3 (see also Supplementary Movie 1). d Trajectories of co-trafficked ICCs indicated

Article Snippet: Anti-myc antibody (9E10), anti-human αL antibody (TS2/4), and anti-human β2 antibody (TS1/18) were purified from hybridomas purchased from ATCC.

Techniques:

Journal: Nature communications

Article Title: The autophagy component LC3 regulates lymphocyte adhesion via LFA1 transport in response to outside-in signaling.

doi: 10.1038/s41467-025-56631-1

Figure Lengend Snippet: Fig. 2 | Importance of ATG8 family proteins for LFA1-dependent lymphocyte adhesion. a Western blotting analysis of LC3b and GABARAP in BAF/LFA1. LC3b- KO: BAF/LFA1 with deletion of LC3b; GABARAP-KO: BAF/LFA1 with deletion of GABARAP. Two independent experiments were performed. b FACS analysis of LFA1 expression in BAF/LFA1 cells. Surface expression of LFA1 was monitored with monoclonal antibodies (Mean Fluorescence Intensity, MFI: αL [No staining, WT, LC3b-KO, GABARAP-KO] = [83.3, 22400, 22300, 26500], β2 [WT, LC3b-KO, GABARAP-KO] = [23300, 16000, 23900]). Total expression of LFA1 was monitored using the SNAP tag protein fused to β2 (MFI: SNAP [No staining, WT, LC3b-KO, GABARAP-KO] = [77.1, 33700, 30300, 39900]). c Adhesion assay. Areas of cell adhesion were monitored by interference reflection microscopy. WT: n = 45; LC3b- KO: n = 45; GABARAP-KO: n = 48. d Accumulation of LFA1 at the contact surface. Surface LFA1 was stained with a dye-conjugated non-blocking monoclonal antibody and adhered to ICAM1-coated dishes in the presence of PMA. The fluorescence intensity of LFA1 at the contact surface was measured by TIRFM. WT: n = 36; LC3b-

Article Snippet: Anti-myc antibody (9E10), anti-human αL antibody (TS2/4), and anti-human β2 antibody (TS1/18) were purified from hybridomas purchased from ATCC.

Techniques: Western Blot, Expressing, Bioprocessing, Fluorescence, Staining, Cell Adhesion Assay, Microscopy, Blocking Assay

Journal: Nature communications

Article Title: The autophagy component LC3 regulates lymphocyte adhesion via LFA1 transport in response to outside-in signaling.

doi: 10.1038/s41467-025-56631-1

Figure Lengend Snippet: Fig. 3 | Outside-in signaling–induced LC3 clustering is driven by AMPK acti- vation. a Role of outside-in signaling in LC3 and LFA1 co-clustering. Merged images of β2-SNAP (green) and CLIP-LC3 (red) in BAF/LFA1 cells are shown. Inside-out stimulation was mediated by PMA and SDF1. Outside-in stimulation was mediated by the following monoclonal antibodies coated on the glass bottom dish: TS1/18, which induces low-affinity dependent outside-in signaling22, and mAb24, which induces high-affinity LFA1-dependent outside-in signaling. Both inside-out and outside-in signaling pathways are activated by ICAM1 + PMA, which induces the binding of lymphocyte LFA1 to ICAM1 in a PMA stimulation-dependent manner. ICAM1-dependent adhesion of BAF/LFA1 was already verified11. Three independent experiments were performed. Scale bar: 5 µm. b Western blotting analysis of the phosphorylation of mTOR and AMPK substrates. S6K: p70 S6 kinase, a substrate of mTOR; ACC: acetyl-CoA carboxylase, a substrate of AMPK; ULK1: Unc-51 like autophagy activating kinase 1. mTOR: n = 4; AMPK: n = 4. c Effects of rapamycin and

Article Snippet: Anti-myc antibody (9E10), anti-human αL antibody (TS2/4), and anti-human β2 antibody (TS1/18) were purified from hybridomas purchased from ATCC.

Techniques: Bioprocessing, Protein-Protein interactions, Binding Assay, Western Blot, Phospho-proteomics

Journal: Nature communications

Article Title: The autophagy component LC3 regulates lymphocyte adhesion via LFA1 transport in response to outside-in signaling.

doi: 10.1038/s41467-025-56631-1

Figure Lengend Snippet: Fig. 4 | The relevance of the non-canonical autophagy in outside-in signaling- dependent LFA1–LC3 co-clustering and lymphocyte adhesion. a Colocalization analysis of LFA1 + LC3+ cluster with a tandem fusion of the evectin-2 pleckstrin- homology (PH) domain (2×PH) fused with GFP. The colocalization indices of LFA1 (β2) with LC3 or 2×PH, and that of LC3 with 2×PH were calculated (n = 13). b Effects of ATG16L1 deletion and expression of ATG16L1 mutants on lymphocyte adhesion. WT: n = 47; ATG16L1-KO: n = 57; ATG16L1-KO expressing GFP-fused ATG16L1 WT (+WT, n = 49), ΔFBD (+ΔFBD, n = 57), or ΔWD (+ΔWD, n = 51) protein. c Effects of

Article Snippet: Anti-myc antibody (9E10), anti-human αL antibody (TS2/4), and anti-human β2 antibody (TS1/18) were purified from hybridomas purchased from ATCC.

Techniques: Expressing

Journal: Signal Transduction and Targeted Therapy

Article Title: Adipocytes orchestrate obesity-related chronic inflammation through β2-microglobulin

doi: 10.1038/s41392-025-02486-3

Figure Lengend Snippet: a – c A transcriptome analysis of mature adipocytes isolated from EpiWAT of C57BL/6J mice on 16-week NCD and HFD. a Network diagram illustrating the interaction among the top 10 immune-related pathways enriched by KEGG analysis of DEGs. The table showing the degree of interaction for each pathway, as assessed by the cumulative interaction scores of the genes within the pathway. b GSEA of DEGs showing two B2M-associated pathways in adipocytes. c Network diagram illustrating the interaction among genes in antigen processing and presentation pathway (blue) and iron uptake and transport pathway (green), with B2M (red) at the intersection. Relative B2m mRNA levels ( d ), protein expressions ( e ) and relative quantification ( f ) in EpiWAT, SVF and purified mature adipocyte fraction from EpiWAT of 16-week NCD- or HFD-fed mice ( n = 3-8). g Representative fluorescence images of B2M (green) and DAPI (blue) staining in EpiWAT sections of NCD-fed (upper) and HFD-fed (below) mice. The right panel quantifies the area-normalized B2M MFI (n = 3). Scale bar = 50 μm. Relative B2m mRNA levels ( h ), protein expressions ( i ) and relative quantification ( j ) in 3T3-L1 adipocytes treated with PA at the indicated dose for 24 h ( n = 4-5). The data are representative as the mean ± standard deviation (SD), with “n” representing the number of biological replicates per experimental group. Significant in d , f , and g was calculated using a two-tailed Student’s t test. Significant in h and j was calculated using one-way ANOVA followed by Tukey’s HSD post hoc test for multiple comparisons. * p < 0.05, ** p < 0.01, *** p < 0.001. B2M β2-microglobulin, DEGs differentially expressed genes, EpiWAT epididymal adipose tissue, HFD high-fat diet, MFI mean fluorescence intensity, NCD normal chow diet, PA palmitic acid, SVF stromal vascular fraction

Article Snippet: ATMs were isolated from the EpiWAT of NCD-fed B2m f/f mice and sorted with magnetic beads (130-110-443, Miltenyi Biotec).

Techniques: Isolation, Quantitative Proteomics, Purification, Fluorescence, Staining, Standard Deviation, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: Adipocytes orchestrate obesity-related chronic inflammation through β2-microglobulin

doi: 10.1038/s41392-025-02486-3

Figure Lengend Snippet: a Study design of diet induced obesity model. b , c Representative photograph of B2m f/f and B2m cKO mice and their respective fat pads. d Body weights ( n = 8). e Weight and proportion of EpiWAT ( n = 8). f Body content (fat, lean, water) analyzed by NMR spectrometer ( n = 6–8). Representative histological images showing H&E staining and Oil Red O staining of indicated sections of HFD-fed B2m f/f and B2m cKO mice ( g ), with quantitation of average adipocyte size of EpiWAT and SAT section ( h ), NAS ( i ), and quantitation of Oil Red O staining ( j ) ( n = 3). Scale bars, 100 μm. k – n Serum levels of leptin, adiponectin, insulin, and glucagon in HFD-fed B2m f/f and B2m cKO mice after 4 hours of fasting ( n = 6). o , p IPGTT (2 g glucose per kg body weight), IPITT (0.75 U insulin per kg body weight) and their respective AUC ( n = 8). q – t Serum levels of TG, TC, HDL and LDL ( n = 8). u Serum levels of TNFα, IFNγ, IL-1β, and IL-6 ( n = 7-8). The data are represented as the mean ± standard deviation (SD), with “n” representing the number of biological replicates per experimental group. Significance in e , f , h , j – n , AUC in o , AUC in p , and q – u was calculated using a two-tailed Student’s t test. Significance in d , o , and p was calculated using two-way ANOVA followed by Tukey’s HSD post hoc test for multiple comparisons. Significance in i was calculated using the Mann-Whitney U test. * p < 0.05, ** p < 0.01, *** p < 0.001. AUC area under the curve, BAT brown adipose tissue, EpiWAT epididymal adipose tissue, HDL high density lipoprotein, HFD high-fat diet, IPGTT intraperitoneal glucose tolerance test, IPITT intraperitoneal insulin tolerance test, LDL low density lipoprotein, NAS Non-alcoholic fatty liver disease activity score, NCD normal chow diet, SAT subcutaneous adipose tissue, TC cholesterol, TG triglyceride

Article Snippet: ATMs were isolated from the EpiWAT of NCD-fed B2m f/f mice and sorted with magnetic beads (130-110-443, Miltenyi Biotec).

Techniques: Staining, Quantitation Assay, Standard Deviation, Two Tailed Test, MANN-WHITNEY, Activity Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: Adipocytes orchestrate obesity-related chronic inflammation through β2-microglobulin

doi: 10.1038/s41392-025-02486-3

Figure Lengend Snippet: a GSEA of antigen processing and presentation pathway between HFD-fed WT mice and B2m cKO mice. Relative mRNA levels of H2-K1 ( b ) and H2-D1 ( c ) in isolated adipocyte fraction of EpiWAT from NCD- or HFD-fed B2m f/f and B2m cKO mice ( n = 4). Protein expressions and relative quantitation of MHC Class I in whole cell lysates ( d , e ) and membrane component ( f , g ) derived from adipocyte fraction isolated from EpiWAT of NCD- or HFD-fed B2m f/f and B2m cKO mice ( n = 3). h Schematic representation of the in vitro co-culture experiment involving primary adipocytes and CD8 + T cells. CD8 + T cells were isolated from EpiWAT of obese B2m f/f mice using a magnetic bead sorting kit and labeled with CFSE before co-culture. Adipocytes were induced from primary adipocyte precursors in SVF and treated with or without PA (2.5 mM) for 24 hours before co-culture. Flow cytometric analysis of proliferation ( i , j ) and CD69 ( k , l ) expression in CD8 + T cells following co-culture with primary adipocytes. ( n = 3). The data are representative as the mean ± standard deviation (SD), with “n” representing the number of biological replicates per experimental group. Significant in b – d , and f was calculated using two-way ANOVA followed by Tukey’s HSD post hoc test for multiple comparisons. Significant in j and l was calculated using multi-way ANOVA followed by Tukey’s HSD post hoc test for multiple comparisons. * p < 0.05, ** p < 0.01, *** p < 0.001. CFSE Carboxyfluorescein Succinimidyl Ester, EpiWAT epididymal adipose tissue, HFD high fat diet, NCD normal chow diet, PA palmitic acid, SVF stromal vascular fraction, WT wild type

Article Snippet: ATMs were isolated from the EpiWAT of NCD-fed B2m f/f mice and sorted with magnetic beads (130-110-443, Miltenyi Biotec).

Techniques: Isolation, Quantitation Assay, Membrane, Derivative Assay, In Vitro, Co-Culture Assay, Labeling, Expressing, Standard Deviation

Journal: Signal Transduction and Targeted Therapy

Article Title: Adipocytes orchestrate obesity-related chronic inflammation through β2-microglobulin

doi: 10.1038/s41392-025-02486-3

Figure Lengend Snippet: a Relative mRNA levels of Fth and Ftl in the adipocyte fraction from EpiWAT of NCD- or HFD-fed B2m f/f and B2m cKO mice ( n = 8). Protein expressions ( b ) and relative quantitation normalized to the internal reference Actin ( c ) of FTH1 and FTL1 in the lysates of mature adipocytes isolated from EpiWAT of NCD- or HFD-fed B2m f/f and B2m cKO mice ( n = 3). d Total iron content, as measured by iron assay kit, in mature adipocytes from EpiWAT in NCD- or HFD-fed B2m f/f and B2m cKO mice ( n = 8). e The correlation of total iron content in ( d ) with EpiWAT proportion (%) ( n = 32). f Co-immunoprecipitation assay using HFE as bait protein showing the interaction between HFE, TFR1, and TFR2 within the membrane fractions of adipocytes isolated from EpiWAT in NCD- or HFD-fed B2m f/f and B2m cKO mice. Relative mRNA levels of Tfr2 ( g ), Hfe ( h ), Tfr1 ( i ), and HAMP ( j ) in the adipocyte fraction isolated from EpiWAT in NCD- or HFD-fed B2m f/f and B2m cKO mice ( n = 7-8). Protein expressions ( k ) and relative quantitation normalized to the internal reference Vinculin in TFR2 ( l ), HFE ( m ), TFR1 ( n ), hepcidin ( o ), and FPN ( p ) in the lysates of adipocyte fraction isolated from EpiWAT in NCD- or HFD-fed B2m f/f and B2m cKO mice ( n = 3). Protein expressions ( q ) and relative quantitation normalized to the internal reference Na,K-ATPase of TFR2 ( r ), HFE ( s ), TFR1 ( t ), and FPN ( u ) in membrane component derived from adipocyte fraction isolated from EpiWAT in NCD- or HFD-fed B2m f/f and B2m cKO mice ( n = 3). The data are represented as the mean ± standard deviation (SD), with “n” representing the number of biological replicates per experimental group. Significant in a , c , d , g – j , l – p , and r – u was calculated using two-way ANOVA followed by Tukey’s HSD post hoc test for multiple comparisons. * p < 0.05, ** p < 0.01, *** p < 0.001.EpiWAT epididymal adipose tissue, FTH ferritin heavy chain, FTL ferritin light chain, HFD high fat diet, HFE Hereditary hemochromatosis protein, NCD normal chow diet, TFR1 transferrin receptor 1, TFR2 transferrin receptor 2, FPN ferroportin

Article Snippet: ATMs were isolated from the EpiWAT of NCD-fed B2m f/f mice and sorted with magnetic beads (130-110-443, Miltenyi Biotec).

Techniques: Quantitation Assay, Isolation, Iron Assay, Co-Immunoprecipitation Assay, Membrane, Derivative Assay, Standard Deviation

Journal: Signal Transduction and Targeted Therapy

Article Title: Adipocytes orchestrate obesity-related chronic inflammation through β2-microglobulin

doi: 10.1038/s41392-025-02486-3

Figure Lengend Snippet: a – g Adipocytes were induced from primary adipocyte precursors in SVF of NCD-fed B2m f/f and B2m cKO mice and treated with or without PA (2.5 mM) for 24 hours. a cell viability ( n = 4). The levels of ROS ( b , c ), lipid peroxides ( d , e ), and Fe 2+ ( f , g ) in adipocytes were assessed using DCFH, C11-BODIPY 581/591 , and FeRhoNox-1 probes, respectively ( n = 3-6). h – m Adipocytes were induced from primary adipocyte precursors in SVF from EpiWAT of NCD-fed B2m f/f and B2m cKO mice and treated with or without PA (2.5 mM) and/or Erastin (2 μM) for 24 hours. The levels of ROS ( h , i ), lipid peroxides ( j , k ), and Fe 2+ ( l , m ) in adipocytes were assessed using DCFH, C11-BODIPY 581/591 , and FeRhoNox-1 probes, respectively ( n = 3-5). n Ferrous ion content in mature adipocytes from NCD- or HFD-fed B2m f/f and B2m cKO mice, measured using an iron content detection kit ( n = 8). o The correlation of ferrous ion content in ( n ) with EpiWAT proportion (%) in NCD- or HFD-fed B2m f/f and B2m cKO mice ( n = 32). Representative images ( p ) and relative quantification ( q ) of ROS, visualized using staining fluorescent probe DHE, in EpiWAT of HFD-fed B2m f/f and B2m cKO mice, DAPI (blue). ( n = 5). Scale bar, 100 μm. Levels of MDA ( r ) and GSH ( s ) in mature adipocytes isolated from EpiWAT in NCD- or HFD-fed B2m f/f and B2m cKO mice ( n = 8). t Representative transmission electron microscopy images of the mitochondria (indicated by red arrow) of adipocytes in EpiWAT of NCD- or HFD-fed B2m f/f and B2m cKO mice. ( n = 5-6). u Flow cytometry analysis of CD86, CD80, and CD11c expressions on ATMs following 24 hours of co-culture with primary adipocytes. ATMs were isolated from EpiWAT of lean B2m f/f mice via F4/80 + magnetic beads. Primary adipocytes were differentiated from adipocyte precursors in SVF from EpiWAT of lean B2m f/f or B2m cKO mice and treated with or without PA (2.5 mM) for 24 hours prior to co-culture ( n = 4). v Representative immunofluorescence staining images of ATMs in EpiWAT of NCD- or HFD-fed B2m f/f and B2m cKO mice, showing perilipin (green), 4-HNE (orange), F4/80 (cyan), CD206 (yellow), CD11c (red), and DAPI (blue). Scale bar, 50 μm. The data are represented as the mean ± standard deviation (SD), with “n” representing the number of biological replicates per experimental group. Significance in q was calculated using a two-tailed Student’s t test. Significant in a , c , e , g , i , k , m , n , r , s , and u was calculated using two-way ANOVA followed by Tukey’s HSD post hoc test for multiple comparisons. * p < 0.05, ** p < 0.01, *** p < 0.001. 4-HNE 4-hydroxynonenal, ATMs adipose tissue macrophages, DHE Dihydroethidium, EpiWAT epididymal adipose tissue, GSH glutathione, HFD high fat diet, MDA malondialdehyde, NCD normal chow diet, PA palmitic acid, ROS reactive oxygen species, SVF stromal vascular fraction

Article Snippet: ATMs were isolated from the EpiWAT of NCD-fed B2m f/f mice and sorted with magnetic beads (130-110-443, Miltenyi Biotec).

Techniques: Quantitative Proteomics, Staining, Isolation, Transmission Assay, Electron Microscopy, Flow Cytometry, Co-Culture Assay, Magnetic Beads, Immunofluorescence, Standard Deviation, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: Adipocytes orchestrate obesity-related chronic inflammation through β2-microglobulin

doi: 10.1038/s41392-025-02486-3

Figure Lengend Snippet: a Overview for AAV9-mediated knockdown of B2M in EpiWAT. b Experiment scheme of male mice was injected with either AAV9-Fabp5-ZsGreen- B2m- shRNA (AAV9- B2m ) or AAV9-Fabp5-ZsGreen (AAV9-null) following six-week HFD feeding and subsequently maintained on the same diet for an additional 6 weeks. c Ex vivo organ fluorescence imaging of EpiWAT, Heart, Liver, Spleen, Lung and Kidney from AAV9- B2m mice and age-matched sham-operated mice at 2 weeks post-injection. d Fluorescence intensity of tissues in c ( n = 3). e Relative B2m mRNA expression in EpiWAT, Heart, Liver, Spleen, Lung and Kidney ( n = 3). Protein expression ( f ) and relative quantification ( g ) of B2M in adipocytes derived from EpiWAT ( n = 3). h Representative photograph of AAV9-null and AAV9- B2m mice. i Body weights ( n = 6). j Weight and proportion of EpiWAT ( n = 6). Representative histological images showing H&E staining of EpiWAT ( k ), with quantitation of average adipocyte size ( l ) ( n = 3). Scale bar, 100 μm. m – o Representative histological images showing H&E staining and Oil Red O staining of liver sections, with NAS ( n ) and relative quantitation of Oil Red O staining ( o ) ( n = 3). Scale bar, 100 μm. p – s IPGTT (2 g glucose per kg body weight), IPITT (0.75 U insulin per kg body weight) and their respective AUC ( n = 6). t Serum levels of TG, TC, HDL and LDL ( n = 3). Relative mRNA levels of inflammatory cytokines ( u ) and adipocytokines ( v ) in EpiWAT ( n = 3). The data are representative as the mean ± standard deviation (SD), with “n” representing the number of biological replicates per experimental group. Significant in d , e , g , j , l , o , q , and s – v was calculated using a two-tailed Student’s t test. Significant in i, p and r was calculated using two-way ANOVA followed by Tukey’s HSD post hoc test for multiple comparisons. Significant in n was calculated using Mann-Whitney U test. * p < 0.05, ** p < 0.01, *** p < 0.001. AAV9 type 9 adeno-associated virus, AUC area under the curve, EpiWAT epididymal adipose tissue, HDL high density lipoprotein, HFD high-fat diet, IPGTT intraperitoneal glucose tolerance test, IPITT intraperitoneal insulin tolerance test, LDL low density lipoprotein, NAS Non-alcoholic fatty liver disease activity score, Sham sham-operated group, TC cholesterol, TG triglyceride

Article Snippet: ATMs were isolated from the EpiWAT of NCD-fed B2m f/f mice and sorted with magnetic beads (130-110-443, Miltenyi Biotec).

Techniques: Knockdown, Injection, shRNA, Ex Vivo, Fluorescence, Imaging, Expressing, Quantitative Proteomics, Derivative Assay, Staining, Quantitation Assay, Standard Deviation, Two Tailed Test, MANN-WHITNEY, Virus, Activity Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: Adipocytes orchestrate obesity-related chronic inflammation through β2-microglobulin

doi: 10.1038/s41392-025-02486-3

Figure Lengend Snippet: KEGG bubble diagram ( a ) and GO bar chart ( b ) displaying selected enrichment pathways of DEGs in SAT from lean and metabolically unhealthy obese patients. c Relative expression of B2m , HLA-A , HLA-B , HLA-C , Fth and Ftl gene in SAT across lean patients ( n = 11), metabolically healthy obese patients ( n = 14) and metabolically unhealthy obese patients ( n = 20). d Correlation analysis of indicated genes and the Mantel test analysis of clinical indicators (BMI, HOMA-IR and mean adipocyte size) and indicated genes among lean and obese-unhealthy patients. A color gradient is utilized to denote Person’s correlation with the value reflecting the precise value of this coefficient. The color of the line indicates the correlation direction in the Mantel test (Mantel’s r. sign), with red indicating a positive correlation and blue signifying a negative correlation. The thickness of the lines reflects the strength of the correlation in the Mantel test (Mantel’s r.abs): thin lines for weak (r < 0.1), standard lines for moderate (0.1 < r < 0.3), and thick lines for strong (r >= 0.3) correlations. The type of line denotes the significance of the Mantel test (Mantel’s p), with solid indicating a significant correlation and dashed signifying a non-significant correlation. e GSEA of DEGs showing significant upregulation of three pathways in SAT from lean patients and metabolically unhealthy obese patients. f Relative expression of B2m in VAT. GSE286454 dataset with lean (n = 4) and obese (n = 3) and GSE283367 ) with lean (n = 70) and obese (n = 43). g – j Protein expressions and relative quantification of B2M, MHC Class I, TFR2, TFR1, FPN, HFE, FTH, FTL and hepcidin in whole cell lysates derived from adipocyte fraction isolated from VAT across lean patients and obese patients ( n = 3). The data are representative as the mean ± standard deviation (SD), with “n” representing the number of biological replicates per experimental group. Significant in f , h , and j was calculated using a two-tailed Student’s t test. Significant in c was calculated using one-way ANOVA followed by Tukey’s HSD post hoc test for multiple comparisons. * p < 0.05, ** p < 0.01, *** p < 0.001. B2M β2-microglobulin, BMI body mass index, DEGs differentially expressed genes, FTH ferritin heavy chain, FTL ferritin light chain, HFE Hereditary hemochromatosis protein, HOMA-IR homeostasis model assessment of insulin resistance, SAT subcutaneous adipose tissue, TFR1 transferrin receptor 1, TFR2 transferrin receptor 2, FPN ferroportin, VAT visceral adipose tissue

Article Snippet: ATMs were isolated from the EpiWAT of NCD-fed B2m f/f mice and sorted with magnetic beads (130-110-443, Miltenyi Biotec).

Techniques: Metabolic Labelling, Expressing, Quantitative Proteomics, Derivative Assay, Isolation, Standard Deviation, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: Adipocytes orchestrate obesity-related chronic inflammation through β2-microglobulin

doi: 10.1038/s41392-025-02486-3

Figure Lengend Snippet: A schematic illustration of the proposed mechanism by which hypertrophic adipocytes induce chronic adipose inflammation and metabolic disorders associated with obesity. Excessive energy stimulates adipocytes to upregulate B2M expression, which not only facilitates the activation and proliferation of CD8 + T cells in adipose tissue through the endogenous antigen presenting pathway, but also promotes iron overload and subsequent ferroptosis of hypertrophic adipocytes via the HFE/B2M-TFR2-Hepcidin-FPN axis, leading to the polarization of ATMs towards M1, thereby accelerating chronic inflammation metabolic disorders. ATMs, adipose tissue macrophages. This figure was created using Adobe Illustrator

Article Snippet: ATMs were isolated from the EpiWAT of NCD-fed B2m f/f mice and sorted with magnetic beads (130-110-443, Miltenyi Biotec).

Techniques: Expressing, Activation Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: Adipocytes orchestrate obesity-related chronic inflammation through β2-microglobulin

doi: 10.1038/s41392-025-02486-3

Figure Lengend Snippet: a – c A transcriptome analysis of mature adipocytes isolated from EpiWAT of C57BL/6J mice on 16-week NCD and HFD. a Network diagram illustrating the interaction among the top 10 immune-related pathways enriched by KEGG analysis of DEGs. The table showing the degree of interaction for each pathway, as assessed by the cumulative interaction scores of the genes within the pathway. b GSEA of DEGs showing two B2M-associated pathways in adipocytes. c Network diagram illustrating the interaction among genes in antigen processing and presentation pathway (blue) and iron uptake and transport pathway (green), with B2M (red) at the intersection. Relative B2m mRNA levels ( d ), protein expressions ( e ) and relative quantification ( f ) in EpiWAT, SVF and purified mature adipocyte fraction from EpiWAT of 16-week NCD- or HFD-fed mice ( n = 3-8). g Representative fluorescence images of B2M (green) and DAPI (blue) staining in EpiWAT sections of NCD-fed (upper) and HFD-fed (below) mice. The right panel quantifies the area-normalized B2M MFI (n = 3). Scale bar = 50 μm. Relative B2m mRNA levels ( h ), protein expressions ( i ) and relative quantification ( j ) in 3T3-L1 adipocytes treated with PA at the indicated dose for 24 h ( n = 4-5). The data are representative as the mean ± standard deviation (SD), with “n” representing the number of biological replicates per experimental group. Significant in d , f , and g was calculated using a two-tailed Student’s t test. Significant in h and j was calculated using one-way ANOVA followed by Tukey’s HSD post hoc test for multiple comparisons. * p < 0.05, ** p < 0.01, *** p < 0.001. B2M β2-microglobulin, DEGs differentially expressed genes, EpiWAT epididymal adipose tissue, HFD high-fat diet, MFI mean fluorescence intensity, NCD normal chow diet, PA palmitic acid, SVF stromal vascular fraction

Article Snippet: CD8 + T cells were isolated from the EpiWAT of HFD-fed B2m f/f mice via magnetic beads (130-104-075, Miltenyi Biotec).

Techniques: Isolation, Quantitative Proteomics, Purification, Fluorescence, Staining, Standard Deviation, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: Adipocytes orchestrate obesity-related chronic inflammation through β2-microglobulin

doi: 10.1038/s41392-025-02486-3

Figure Lengend Snippet: a Study design of diet induced obesity model. b , c Representative photograph of B2m f/f and B2m cKO mice and their respective fat pads. d Body weights ( n = 8). e Weight and proportion of EpiWAT ( n = 8). f Body content (fat, lean, water) analyzed by NMR spectrometer ( n = 6–8). Representative histological images showing H&E staining and Oil Red O staining of indicated sections of HFD-fed B2m f/f and B2m cKO mice ( g ), with quantitation of average adipocyte size of EpiWAT and SAT section ( h ), NAS ( i ), and quantitation of Oil Red O staining ( j ) ( n = 3). Scale bars, 100 μm. k – n Serum levels of leptin, adiponectin, insulin, and glucagon in HFD-fed B2m f/f and B2m cKO mice after 4 hours of fasting ( n = 6). o , p IPGTT (2 g glucose per kg body weight), IPITT (0.75 U insulin per kg body weight) and their respective AUC ( n = 8). q – t Serum levels of TG, TC, HDL and LDL ( n = 8). u Serum levels of TNFα, IFNγ, IL-1β, and IL-6 ( n = 7-8). The data are represented as the mean ± standard deviation (SD), with “n” representing the number of biological replicates per experimental group. Significance in e , f , h , j – n , AUC in o , AUC in p , and q – u was calculated using a two-tailed Student’s t test. Significance in d , o , and p was calculated using two-way ANOVA followed by Tukey’s HSD post hoc test for multiple comparisons. Significance in i was calculated using the Mann-Whitney U test. * p < 0.05, ** p < 0.01, *** p < 0.001. AUC area under the curve, BAT brown adipose tissue, EpiWAT epididymal adipose tissue, HDL high density lipoprotein, HFD high-fat diet, IPGTT intraperitoneal glucose tolerance test, IPITT intraperitoneal insulin tolerance test, LDL low density lipoprotein, NAS Non-alcoholic fatty liver disease activity score, NCD normal chow diet, SAT subcutaneous adipose tissue, TC cholesterol, TG triglyceride

Article Snippet: CD8 + T cells were isolated from the EpiWAT of HFD-fed B2m f/f mice via magnetic beads (130-104-075, Miltenyi Biotec).

Techniques: Staining, Quantitation Assay, Standard Deviation, Two Tailed Test, MANN-WHITNEY, Activity Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: Adipocytes orchestrate obesity-related chronic inflammation through β2-microglobulin

doi: 10.1038/s41392-025-02486-3

Figure Lengend Snippet: a GSEA of antigen processing and presentation pathway between HFD-fed WT mice and B2m cKO mice. Relative mRNA levels of H2-K1 ( b ) and H2-D1 ( c ) in isolated adipocyte fraction of EpiWAT from NCD- or HFD-fed B2m f/f and B2m cKO mice ( n = 4). Protein expressions and relative quantitation of MHC Class I in whole cell lysates ( d , e ) and membrane component ( f , g ) derived from adipocyte fraction isolated from EpiWAT of NCD- or HFD-fed B2m f/f and B2m cKO mice ( n = 3). h Schematic representation of the in vitro co-culture experiment involving primary adipocytes and CD8 + T cells. CD8 + T cells were isolated from EpiWAT of obese B2m f/f mice using a magnetic bead sorting kit and labeled with CFSE before co-culture. Adipocytes were induced from primary adipocyte precursors in SVF and treated with or without PA (2.5 mM) for 24 hours before co-culture. Flow cytometric analysis of proliferation ( i , j ) and CD69 ( k , l ) expression in CD8 + T cells following co-culture with primary adipocytes. ( n = 3). The data are representative as the mean ± standard deviation (SD), with “n” representing the number of biological replicates per experimental group. Significant in b – d , and f was calculated using two-way ANOVA followed by Tukey’s HSD post hoc test for multiple comparisons. Significant in j and l was calculated using multi-way ANOVA followed by Tukey’s HSD post hoc test for multiple comparisons. * p < 0.05, ** p < 0.01, *** p < 0.001. CFSE Carboxyfluorescein Succinimidyl Ester, EpiWAT epididymal adipose tissue, HFD high fat diet, NCD normal chow diet, PA palmitic acid, SVF stromal vascular fraction, WT wild type

Article Snippet: CD8 + T cells were isolated from the EpiWAT of HFD-fed B2m f/f mice via magnetic beads (130-104-075, Miltenyi Biotec).

Techniques: Isolation, Quantitation Assay, Membrane, Derivative Assay, In Vitro, Co-Culture Assay, Labeling, Expressing, Standard Deviation

Journal: Signal Transduction and Targeted Therapy

Article Title: Adipocytes orchestrate obesity-related chronic inflammation through β2-microglobulin

doi: 10.1038/s41392-025-02486-3

Figure Lengend Snippet: a Relative mRNA levels of Fth and Ftl in the adipocyte fraction from EpiWAT of NCD- or HFD-fed B2m f/f and B2m cKO mice ( n = 8). Protein expressions ( b ) and relative quantitation normalized to the internal reference Actin ( c ) of FTH1 and FTL1 in the lysates of mature adipocytes isolated from EpiWAT of NCD- or HFD-fed B2m f/f and B2m cKO mice ( n = 3). d Total iron content, as measured by iron assay kit, in mature adipocytes from EpiWAT in NCD- or HFD-fed B2m f/f and B2m cKO mice ( n = 8). e The correlation of total iron content in ( d ) with EpiWAT proportion (%) ( n = 32). f Co-immunoprecipitation assay using HFE as bait protein showing the interaction between HFE, TFR1, and TFR2 within the membrane fractions of adipocytes isolated from EpiWAT in NCD- or HFD-fed B2m f/f and B2m cKO mice. Relative mRNA levels of Tfr2 ( g ), Hfe ( h ), Tfr1 ( i ), and HAMP ( j ) in the adipocyte fraction isolated from EpiWAT in NCD- or HFD-fed B2m f/f and B2m cKO mice ( n = 7-8). Protein expressions ( k ) and relative quantitation normalized to the internal reference Vinculin in TFR2 ( l ), HFE ( m ), TFR1 ( n ), hepcidin ( o ), and FPN ( p ) in the lysates of adipocyte fraction isolated from EpiWAT in NCD- or HFD-fed B2m f/f and B2m cKO mice ( n = 3). Protein expressions ( q ) and relative quantitation normalized to the internal reference Na,K-ATPase of TFR2 ( r ), HFE ( s ), TFR1 ( t ), and FPN ( u ) in membrane component derived from adipocyte fraction isolated from EpiWAT in NCD- or HFD-fed B2m f/f and B2m cKO mice ( n = 3). The data are represented as the mean ± standard deviation (SD), with “n” representing the number of biological replicates per experimental group. Significant in a , c , d , g – j , l – p , and r – u was calculated using two-way ANOVA followed by Tukey’s HSD post hoc test for multiple comparisons. * p < 0.05, ** p < 0.01, *** p < 0.001.EpiWAT epididymal adipose tissue, FTH ferritin heavy chain, FTL ferritin light chain, HFD high fat diet, HFE Hereditary hemochromatosis protein, NCD normal chow diet, TFR1 transferrin receptor 1, TFR2 transferrin receptor 2, FPN ferroportin

Article Snippet: CD8 + T cells were isolated from the EpiWAT of HFD-fed B2m f/f mice via magnetic beads (130-104-075, Miltenyi Biotec).

Techniques: Quantitation Assay, Isolation, Iron Assay, Co-Immunoprecipitation Assay, Membrane, Derivative Assay, Standard Deviation

Journal: Signal Transduction and Targeted Therapy

Article Title: Adipocytes orchestrate obesity-related chronic inflammation through β2-microglobulin

doi: 10.1038/s41392-025-02486-3

Figure Lengend Snippet: a – g Adipocytes were induced from primary adipocyte precursors in SVF of NCD-fed B2m f/f and B2m cKO mice and treated with or without PA (2.5 mM) for 24 hours. a cell viability ( n = 4). The levels of ROS ( b , c ), lipid peroxides ( d , e ), and Fe 2+ ( f , g ) in adipocytes were assessed using DCFH, C11-BODIPY 581/591 , and FeRhoNox-1 probes, respectively ( n = 3-6). h – m Adipocytes were induced from primary adipocyte precursors in SVF from EpiWAT of NCD-fed B2m f/f and B2m cKO mice and treated with or without PA (2.5 mM) and/or Erastin (2 μM) for 24 hours. The levels of ROS ( h , i ), lipid peroxides ( j , k ), and Fe 2+ ( l , m ) in adipocytes were assessed using DCFH, C11-BODIPY 581/591 , and FeRhoNox-1 probes, respectively ( n = 3-5). n Ferrous ion content in mature adipocytes from NCD- or HFD-fed B2m f/f and B2m cKO mice, measured using an iron content detection kit ( n = 8). o The correlation of ferrous ion content in ( n ) with EpiWAT proportion (%) in NCD- or HFD-fed B2m f/f and B2m cKO mice ( n = 32). Representative images ( p ) and relative quantification ( q ) of ROS, visualized using staining fluorescent probe DHE, in EpiWAT of HFD-fed B2m f/f and B2m cKO mice, DAPI (blue). ( n = 5). Scale bar, 100 μm. Levels of MDA ( r ) and GSH ( s ) in mature adipocytes isolated from EpiWAT in NCD- or HFD-fed B2m f/f and B2m cKO mice ( n = 8). t Representative transmission electron microscopy images of the mitochondria (indicated by red arrow) of adipocytes in EpiWAT of NCD- or HFD-fed B2m f/f and B2m cKO mice. ( n = 5-6). u Flow cytometry analysis of CD86, CD80, and CD11c expressions on ATMs following 24 hours of co-culture with primary adipocytes. ATMs were isolated from EpiWAT of lean B2m f/f mice via F4/80 + magnetic beads. Primary adipocytes were differentiated from adipocyte precursors in SVF from EpiWAT of lean B2m f/f or B2m cKO mice and treated with or without PA (2.5 mM) for 24 hours prior to co-culture ( n = 4). v Representative immunofluorescence staining images of ATMs in EpiWAT of NCD- or HFD-fed B2m f/f and B2m cKO mice, showing perilipin (green), 4-HNE (orange), F4/80 (cyan), CD206 (yellow), CD11c (red), and DAPI (blue). Scale bar, 50 μm. The data are represented as the mean ± standard deviation (SD), with “n” representing the number of biological replicates per experimental group. Significance in q was calculated using a two-tailed Student’s t test. Significant in a , c , e , g , i , k , m , n , r , s , and u was calculated using two-way ANOVA followed by Tukey’s HSD post hoc test for multiple comparisons. * p < 0.05, ** p < 0.01, *** p < 0.001. 4-HNE 4-hydroxynonenal, ATMs adipose tissue macrophages, DHE Dihydroethidium, EpiWAT epididymal adipose tissue, GSH glutathione, HFD high fat diet, MDA malondialdehyde, NCD normal chow diet, PA palmitic acid, ROS reactive oxygen species, SVF stromal vascular fraction

Article Snippet: CD8 + T cells were isolated from the EpiWAT of HFD-fed B2m f/f mice via magnetic beads (130-104-075, Miltenyi Biotec).

Techniques: Quantitative Proteomics, Staining, Isolation, Transmission Assay, Electron Microscopy, Flow Cytometry, Co-Culture Assay, Magnetic Beads, Immunofluorescence, Standard Deviation, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: Adipocytes orchestrate obesity-related chronic inflammation through β2-microglobulin

doi: 10.1038/s41392-025-02486-3

Figure Lengend Snippet: a Overview for AAV9-mediated knockdown of B2M in EpiWAT. b Experiment scheme of male mice was injected with either AAV9-Fabp5-ZsGreen- B2m- shRNA (AAV9- B2m ) or AAV9-Fabp5-ZsGreen (AAV9-null) following six-week HFD feeding and subsequently maintained on the same diet for an additional 6 weeks. c Ex vivo organ fluorescence imaging of EpiWAT, Heart, Liver, Spleen, Lung and Kidney from AAV9- B2m mice and age-matched sham-operated mice at 2 weeks post-injection. d Fluorescence intensity of tissues in c ( n = 3). e Relative B2m mRNA expression in EpiWAT, Heart, Liver, Spleen, Lung and Kidney ( n = 3). Protein expression ( f ) and relative quantification ( g ) of B2M in adipocytes derived from EpiWAT ( n = 3). h Representative photograph of AAV9-null and AAV9- B2m mice. i Body weights ( n = 6). j Weight and proportion of EpiWAT ( n = 6). Representative histological images showing H&E staining of EpiWAT ( k ), with quantitation of average adipocyte size ( l ) ( n = 3). Scale bar, 100 μm. m – o Representative histological images showing H&E staining and Oil Red O staining of liver sections, with NAS ( n ) and relative quantitation of Oil Red O staining ( o ) ( n = 3). Scale bar, 100 μm. p – s IPGTT (2 g glucose per kg body weight), IPITT (0.75 U insulin per kg body weight) and their respective AUC ( n = 6). t Serum levels of TG, TC, HDL and LDL ( n = 3). Relative mRNA levels of inflammatory cytokines ( u ) and adipocytokines ( v ) in EpiWAT ( n = 3). The data are representative as the mean ± standard deviation (SD), with “n” representing the number of biological replicates per experimental group. Significant in d , e , g , j , l , o , q , and s – v was calculated using a two-tailed Student’s t test. Significant in i, p and r was calculated using two-way ANOVA followed by Tukey’s HSD post hoc test for multiple comparisons. Significant in n was calculated using Mann-Whitney U test. * p < 0.05, ** p < 0.01, *** p < 0.001. AAV9 type 9 adeno-associated virus, AUC area under the curve, EpiWAT epididymal adipose tissue, HDL high density lipoprotein, HFD high-fat diet, IPGTT intraperitoneal glucose tolerance test, IPITT intraperitoneal insulin tolerance test, LDL low density lipoprotein, NAS Non-alcoholic fatty liver disease activity score, Sham sham-operated group, TC cholesterol, TG triglyceride

Article Snippet: CD8 + T cells were isolated from the EpiWAT of HFD-fed B2m f/f mice via magnetic beads (130-104-075, Miltenyi Biotec).

Techniques: Knockdown, Injection, shRNA, Ex Vivo, Fluorescence, Imaging, Expressing, Quantitative Proteomics, Derivative Assay, Staining, Quantitation Assay, Standard Deviation, Two Tailed Test, MANN-WHITNEY, Virus, Activity Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: Adipocytes orchestrate obesity-related chronic inflammation through β2-microglobulin

doi: 10.1038/s41392-025-02486-3

Figure Lengend Snippet: KEGG bubble diagram ( a ) and GO bar chart ( b ) displaying selected enrichment pathways of DEGs in SAT from lean and metabolically unhealthy obese patients. c Relative expression of B2m , HLA-A , HLA-B , HLA-C , Fth and Ftl gene in SAT across lean patients ( n = 11), metabolically healthy obese patients ( n = 14) and metabolically unhealthy obese patients ( n = 20). d Correlation analysis of indicated genes and the Mantel test analysis of clinical indicators (BMI, HOMA-IR and mean adipocyte size) and indicated genes among lean and obese-unhealthy patients. A color gradient is utilized to denote Person’s correlation with the value reflecting the precise value of this coefficient. The color of the line indicates the correlation direction in the Mantel test (Mantel’s r. sign), with red indicating a positive correlation and blue signifying a negative correlation. The thickness of the lines reflects the strength of the correlation in the Mantel test (Mantel’s r.abs): thin lines for weak (r < 0.1), standard lines for moderate (0.1 < r < 0.3), and thick lines for strong (r >= 0.3) correlations. The type of line denotes the significance of the Mantel test (Mantel’s p), with solid indicating a significant correlation and dashed signifying a non-significant correlation. e GSEA of DEGs showing significant upregulation of three pathways in SAT from lean patients and metabolically unhealthy obese patients. f Relative expression of B2m in VAT. GSE286454 dataset with lean (n = 4) and obese (n = 3) and GSE283367 ) with lean (n = 70) and obese (n = 43). g – j Protein expressions and relative quantification of B2M, MHC Class I, TFR2, TFR1, FPN, HFE, FTH, FTL and hepcidin in whole cell lysates derived from adipocyte fraction isolated from VAT across lean patients and obese patients ( n = 3). The data are representative as the mean ± standard deviation (SD), with “n” representing the number of biological replicates per experimental group. Significant in f , h , and j was calculated using a two-tailed Student’s t test. Significant in c was calculated using one-way ANOVA followed by Tukey’s HSD post hoc test for multiple comparisons. * p < 0.05, ** p < 0.01, *** p < 0.001. B2M β2-microglobulin, BMI body mass index, DEGs differentially expressed genes, FTH ferritin heavy chain, FTL ferritin light chain, HFE Hereditary hemochromatosis protein, HOMA-IR homeostasis model assessment of insulin resistance, SAT subcutaneous adipose tissue, TFR1 transferrin receptor 1, TFR2 transferrin receptor 2, FPN ferroportin, VAT visceral adipose tissue

Article Snippet: CD8 + T cells were isolated from the EpiWAT of HFD-fed B2m f/f mice via magnetic beads (130-104-075, Miltenyi Biotec).

Techniques: Metabolic Labelling, Expressing, Quantitative Proteomics, Derivative Assay, Isolation, Standard Deviation, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: Adipocytes orchestrate obesity-related chronic inflammation through β2-microglobulin

doi: 10.1038/s41392-025-02486-3

Figure Lengend Snippet: A schematic illustration of the proposed mechanism by which hypertrophic adipocytes induce chronic adipose inflammation and metabolic disorders associated with obesity. Excessive energy stimulates adipocytes to upregulate B2M expression, which not only facilitates the activation and proliferation of CD8 + T cells in adipose tissue through the endogenous antigen presenting pathway, but also promotes iron overload and subsequent ferroptosis of hypertrophic adipocytes via the HFE/B2M-TFR2-Hepcidin-FPN axis, leading to the polarization of ATMs towards M1, thereby accelerating chronic inflammation metabolic disorders. ATMs, adipose tissue macrophages. This figure was created using Adobe Illustrator

Article Snippet: CD8 + T cells were isolated from the EpiWAT of HFD-fed B2m f/f mice via magnetic beads (130-104-075, Miltenyi Biotec).

Techniques: Expressing, Activation Assay