Journal: bioRxiv

Article Title: Endothelial FABP4 constitutes the majority of basal circulating hormone levels and regulates lipolysis-driven insulin secretion

doi: 10.1101/2022.10.13.511807

Figure Lengend Snippet: ( A ) Left panel: Representative immunoblot of FABP4 expression in perirenal adipose tissue of WT, Adipo-KO, Endo-KO, and Total-KO mice. Right panel: Quantification of FABP4 relative to β-tubulin signal.***p<0.001, **p<0.01. ( B ) FABP4 immunostaining in perigonadal adipose tissue from WT, Adipo-KO, Endo-KO, and Total-KO mice. 40X magnification. ( C ) Left panel: Immunoblot of FABP4 expression in isolated PGWAT adipocytes of WT, Adipo-KO, Endo-KO, and Total-KO mice. Right panel: Quantification of FABP4 relative to β-tubulin signal.*p<0.05, **p<0.01. n=1 for Total-KO. Data were analyzed by one-way ANOVA followed by Tukey’s multiple comparison test and are presented as mean ± SEM. ND = No signal detected.

Article Snippet: For in-house FABP4 ELISA we used anti-FABP4 antibodies produced for the Hotamışlıgil lab by the Dana Farber Antibody Core (clone 351.4.6H7.G3.G9 for capture, HRP-tagged clone 351.4.5E1.H3 for detection) and recombinant human FABP4 as a standard (R&D Systems, cat# DY3150-05).

Techniques: Western Blot, Expressing, Immunostaining, Isolation, Comparison

Journal: bioRxiv

Article Title: Endothelial FABP4 constitutes the majority of basal circulating hormone levels and regulates lipolysis-driven insulin secretion

doi: 10.1101/2022.10.13.511807

Figure Lengend Snippet: ( A ) Left panel: Immunoblot of FABP4 expression in isolated liver endothelial cells of WT, Adipo-KO, and Endo-KO mice. Right panel: Quantification of FABP4 relative to β-tubulin signal. ( B ) FABP4 immunostaining in liver from WT, Adipo-KO, Endo-KO, and Myeloid-KO mice. 20X magnification. ND = No signal detected.

Article Snippet: For in-house FABP4 ELISA we used anti-FABP4 antibodies produced for the Hotamışlıgil lab by the Dana Farber Antibody Core (clone 351.4.6H7.G3.G9 for capture, HRP-tagged clone 351.4.5E1.H3 for detection) and recombinant human FABP4 as a standard (R&D Systems, cat# DY3150-05).

Techniques: Western Blot, Expressing, Isolation, Immunostaining

Journal: bioRxiv

Article Title: Endothelial FABP4 constitutes the majority of basal circulating hormone levels and regulates lipolysis-driven insulin secretion

doi: 10.1101/2022.10.13.511807

Figure Lengend Snippet: ( A) Top panel: Plasma FABP4 levels in WT, Adipo-KO, Endo-KO, and Total-KO lean male mice, after 6h daytime food withdrawal. Data are pooled from samples from 9 experiments. ****p<0.0001, **p<0.01. Bottom panel: Immunoblots of perirenal and mesenteric adipose FABP4 protein expression (same images as in and S2A), for comparison with plasma levels. ( B ) Plasma FABP4 levels in 6h WT vs. Myeloid-KO mice. Data are pooled from samples from 2 experiments. ( C ) Plasma FABP5 levels in WT, Adipo-KO, Endo-KO and Total-KO mice. *p<0.05. Data in 3A, 3C, and 3D were analyzed by one-way ANOVA, followed by Tukey’s multiple comparison test. Data in 3B were analyzed by unpaired t-test. Data are presented as mean ± SEM. ND: No signal detected.

Article Snippet: For in-house FABP4 ELISA we used anti-FABP4 antibodies produced for the Hotamışlıgil lab by the Dana Farber Antibody Core (clone 351.4.6H7.G3.G9 for capture, HRP-tagged clone 351.4.5E1.H3 for detection) and recombinant human FABP4 as a standard (R&D Systems, cat# DY3150-05).

Techniques: Clinical Proteomics, Western Blot, Expressing, Comparison

Journal: bioRxiv

Article Title: Endothelial FABP4 constitutes the majority of basal circulating hormone levels and regulates lipolysis-driven insulin secretion

doi: 10.1101/2022.10.13.511807

Figure Lengend Snippet: ( A ) Plasma FABP4, ( B ) Non-esterified fatty acid (NEFA), and ( C ) glycerol responses to 10mg/kg isoproterenol-induced lipolysis in WT, Adipo-KO, Endo-KO, and Total-KO male mice. FABP4 responses are presented as induction over baseline. Data are pooled from 6 separate experiments. WT n=52, Adipo-KO n=39, Endo-KO n=35, Total-KO n=8 for , n=15 for , . ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05 vs. WT; °°p<0.01 vs. Adipo-KO, °p<0.05 vs. Adipo-KO. ( D ) NEFA, ( E ) glycerol, and ( F ) FABP4 responses to forskolin (FSK)-induced lipolysis in perigonadal adipose explants from male WT, Adipo-KO, and Endo-KO mice. Data are normalized to amount of adipose tissue per culture well. n=4 mice/group, 3 replicates per mouse. ****p<0.0001, ***p<0.001 vs. Adipo-KO. Data were analyzed by repeated measures two-way ANOVA followed by Tukey’s multiple comparison test and are presented as mean ± SEM. ND: No signal detected.

Article Snippet: For in-house FABP4 ELISA we used anti-FABP4 antibodies produced for the Hotamışlıgil lab by the Dana Farber Antibody Core (clone 351.4.6H7.G3.G9 for capture, HRP-tagged clone 351.4.5E1.H3 for detection) and recombinant human FABP4 as a standard (R&D Systems, cat# DY3150-05).

Techniques: Clinical Proteomics, Comparison

Journal: bioRxiv

Article Title: Endothelial FABP4 constitutes the majority of basal circulating hormone levels and regulates lipolysis-driven insulin secretion

doi: 10.1101/2022.10.13.511807

Figure Lengend Snippet: ( A ) Plasma FABP4 responses to 10mg/kg isoproterenol-induced lipolysis in WT and Myeloid FABP4-KO mice, n=8/group, and ( B ) in WT vs. mice with deletion of FABP4 in both adipocytes and endothelial cells (Adipo Endo-KO). n=6/group. ****p<0.0001, **p<0.01. Data were analyzed by repeated measures two-way ANOVA followed by Tukey’s multiple comparison test and are presented as mean ± SEM.

Article Snippet: For in-house FABP4 ELISA we used anti-FABP4 antibodies produced for the Hotamışlıgil lab by the Dana Farber Antibody Core (clone 351.4.6H7.G3.G9 for capture, HRP-tagged clone 351.4.5E1.H3 for detection) and recombinant human FABP4 as a standard (R&D Systems, cat# DY3150-05).

Techniques: Clinical Proteomics, Comparison

Journal: bioRxiv

Article Title: Endothelial FABP4 constitutes the majority of basal circulating hormone levels and regulates lipolysis-driven insulin secretion

doi: 10.1101/2022.10.13.511807

Figure Lengend Snippet: ( A ) 4-hour conditioned media FABP4 levels from CD31-isolated liver, heart, and lung endothelial cells of WT, Adipo--KO, and Endo-KO mice. FABP4 levels are normalized to total cellular protein levels. *p<0.05. ( B ) FABP5 levels in CD31-isolated liver endothelial cell 24-hour conditioned media and (C ) lysates of WT and Endo-KO mice. Data in 6A were analyzed by one-way ANOVA, followed by Tukey’s multiple comparison test and are presented as mean ± SEM. ND: No signal detected.

Article Snippet: For in-house FABP4 ELISA we used anti-FABP4 antibodies produced for the Hotamışlıgil lab by the Dana Farber Antibody Core (clone 351.4.6H7.G3.G9 for capture, HRP-tagged clone 351.4.5E1.H3 for detection) and recombinant human FABP4 as a standard (R&D Systems, cat# DY3150-05).

Techniques: Isolation, Comparison

Journal: bioRxiv

Article Title: Endothelial FABP4 constitutes the majority of basal circulating hormone levels and regulates lipolysis-driven insulin secretion

doi: 10.1101/2022.10.13.511807

Figure Lengend Snippet: ( A ) FABP4 in HUVEC lysates normalized to total cellular protein ( B ) and in 5-hour conditioned media normalized to total cellular protein at days 3 through 14 post-seeding. Pool of 2 experiments. *p<0.05, **p<0.01. ( C ) Time-course of cumulative FABP4 levels in media of day 7 HUVECs. n=4/time point. Inset: Media lactate dehydrogenase (LDH) levels during the same time course. n=4. ( D ) Effects of increasing doses of the ER-Golgi pathway inhibitor, brefeldin A (BFA) on FABP4 and endothelin-1 (ET-1) secretion from day 11 HUVECs. n=3 per BFA dose. *p<0.05. ( E ) Effects of increasing doses of forskolin (FSK) on FABP4 secretion in HUVECs vs. 3T3-L1 adipocytes. n=3 per FSK dose. *p<0.05, **p<0.01 vs. HUVEC. ( F ) Effects of increasing doses of FSK on FABP4 and Von Willibrand Factor (vWF) secretion from day 11 HUVECs. n=3 per FSK dose. *p<0.05, **p<0.01. 7A-C were analyzed by one-way ANOVA followed by Tukey’s multiple comparison test. 7D-F were analyzed by repeated measures 2-way ANOVA with Geisser-Greenhouse correction, followed by Dunnett’s (D,F) or Sidak’s (E) multiple comparison test. Data are presented as mean ± SEM.

Article Snippet: For in-house FABP4 ELISA we used anti-FABP4 antibodies produced for the Hotamışlıgil lab by the Dana Farber Antibody Core (clone 351.4.6H7.G3.G9 for capture, HRP-tagged clone 351.4.5E1.H3 for detection) and recombinant human FABP4 as a standard (R&D Systems, cat# DY3150-05).

Techniques: Comparison

Journal: bioRxiv

Article Title: Endothelial FABP4 constitutes the majority of basal circulating hormone levels and regulates lipolysis-driven insulin secretion

doi: 10.1101/2022.10.13.511807

Figure Lengend Snippet: ( A ) Plasma insulin responses to 10mg/kg isoproterenol-induced lipolysis in WT, Adipo-KO, Endo-KO, and Total-KO mice. Data are pooled from 6 experiments. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05 vs. WT. °°°°p<0.0001, °°°p<0.001, °°p<0.01, °p<0.05 vs. Adipo-KO. WT, n=51; Adipo-KO, n=39; Endo-KO, n=34; Total-KO, n=15. ( B ) Plasma insulin responses to 10mg/kg isoproterenol-induced lipolysis in WT vs. Myeloid-KO mice. n=8/group. ( C ) Plasma FAPB4 and ( D ) insulin responses in WT and FABP4-KO mice injected with PBS or 7ug of FABP4 prior to induction of lipolysis with 10mg/kg isoproterenol. n=8/group. ***p<0.0005, **p<0.001, *p<0.05 vs. WT. Data were analyzed by repeated measures 2-way ANOVA with Geisser-Greenhouse correction, followed by Sidak’s (C) or Tukey’s (A,D) multiple comparison test. Data are presented as mean ± SEM. ND: No signal detected.

Article Snippet: For in-house FABP4 ELISA we used anti-FABP4 antibodies produced for the Hotamışlıgil lab by the Dana Farber Antibody Core (clone 351.4.6H7.G3.G9 for capture, HRP-tagged clone 351.4.5E1.H3 for detection) and recombinant human FABP4 as a standard (R&D Systems, cat# DY3150-05).

Techniques: Clinical Proteomics, Injection, Comparison

Journal: bioRxiv

Article Title: Endothelial FABP4 constitutes the majority of basal circulating hormone levels and regulates lipolysis-driven insulin secretion

doi: 10.1101/2022.10.13.511807

Figure Lengend Snippet: ( A ) Upper panel: FABP4 immunostaining of pancreas of WT, Adipo-KO, Endo-KO and Myeloid-KO mice. Islets are encircled by black dotted lines. 40X magnification. Lower panel: 2X enlargement of boxed area in upper panel. ( B ) Insulin immunostaining of pancreas from WT, Adipo-KO, Endo-KO, and Total-KO mice, 40X magnification, and ( C ) quantification of insulin-positive area. ( D ) Insulin secretion from isolated islets of WT, Adipo-KO, Endo-KO and Total-KO mice in response to low glucose (2.8mM), high glucose (16.7mM), high glucose + KCl (30mM). *p<0.05. ( E ) Fold-increase in insulin secretion induced by HG + FSK (10uM) over HG. Data in D were analyzed by unpaired t-test. Data in C and E were analyzed by one-way ANOVA followed by Tukey’s multiple comparison test. Data are presented as mean ± SEM.

Article Snippet: For in-house FABP4 ELISA we used anti-FABP4 antibodies produced for the Hotamışlıgil lab by the Dana Farber Antibody Core (clone 351.4.6H7.G3.G9 for capture, HRP-tagged clone 351.4.5E1.H3 for detection) and recombinant human FABP4 as a standard (R&D Systems, cat# DY3150-05).

Techniques: Immunostaining, Isolation, Comparison

Journal: bioRxiv

Article Title: Endothelial FABP4 constitutes the majority of basal circulating hormone levels and regulates lipolysis-driven insulin secretion

doi: 10.1101/2022.10.13.511807

Figure Lengend Snippet: ( A) The mouse FABP4 gene was first converted to the the human FABP4 open reading frame by substituting the 11 amino acids differing between mouse and human FABP4 located in exons 2 and 3. ( B ) Exon 2 was flanked at the 3’ end by a FRT-neomycin-FRT-loxP cassette and by a single lox P site at the 5’ end. This distal lox P site was positioned upstream of exon 2 within the intron 1 sequences. The model was generated by homologous recombination in embryonic stem cells. The FRT-flanked selection cassette was removed in vivo by crossing with Flp-recombinase-expressing mice. Hatched rectangles represent FABP4 coding sequences. Gray rectangles indicate non-coding exon portions. Solid lines represent chromosome sequences. The 11 murine/human amino acid substitutions are represented by stars.

Article Snippet: For in-house FABP4 ELISA we used anti-FABP4 antibodies produced for the Hotamışlıgil lab by the Dana Farber Antibody Core (clone 351.4.6H7.G3.G9 for capture, HRP-tagged clone 351.4.5E1.H3 for detection) and recombinant human FABP4 as a standard (R&D Systems, cat# DY3150-05).

Techniques: Generated, Homologous Recombination, Selection, In Vivo, Expressing

Journal: Scientific Reports

Article Title: Adipocyte Fatty-Acid Binding Protein is Overexpressed in Cirrhosis and Correlates with Clinical Outcomes

doi: 10.1038/s41598-017-01709-0

Figure Lengend Snippet: Plasma levels of FABPs according to the presence or absence of complications of cirrhosis at inclusion.

Article Snippet: L-FABP1, I-FABP2 and A-FABP4 were measured with the human L-FABP1 ELISA kit (Hycult Biotech; Uden, The Neatherlands), the human I-FABP2 ELISA kit (Hycult Biotech; Uden, The Neatherlands) and the Quantikine ELISA human A-FABP4 kit (R&D Systems, Minneapolis, USA), respectively.

Techniques: Clinical Proteomics

Journal: Scientific Reports

Article Title: Adipocyte Fatty-Acid Binding Protein is Overexpressed in Cirrhosis and Correlates with Clinical Outcomes

doi: 10.1038/s41598-017-01709-0

Figure Lengend Snippet: Cox regression analysis of 90-day transplant-free survival.

Article Snippet: L-FABP1, I-FABP2 and A-FABP4 were measured with the human L-FABP1 ELISA kit (Hycult Biotech; Uden, The Neatherlands), the human I-FABP2 ELISA kit (Hycult Biotech; Uden, The Neatherlands) and the Quantikine ELISA human A-FABP4 kit (R&D Systems, Minneapolis, USA), respectively.

Techniques:

Journal: Cytotherapy

Article Title: Assay validation for the assessment of adipogenesis of multipotential stromal cells—a direct comparison of four different methods

doi: 10.1016/j.jcyt.2012.07.001

Figure Lengend Snippet: Monitoring adipogenic progression of MSCs and fibroblasts with the use of q-PCR. MSCs and fibroblasts were cultured in adipogenic medium for 21 days. Expression of (A) PPAR-γ and (B) FABP4 was determined on days 0, 3, 7, 14 and 21. Relative levels of gene expression were normalized to reference gene GAPDH and displayed as fold increase over day 0. Each data point represents the mean of three replicates.

Article Snippet: FABP4 protein was detected with the use of a purified polyclonal goat anti-human FABP4 antibody (IgG, R & D Systems, Abingdon, UK) at 1:20 dilution in PBS.

Techniques: Cell Culture, Expressing, Gene Expression

Journal: Cytotherapy

Article Title: Assay validation for the assessment of adipogenesis of multipotential stromal cells—a direct comparison of four different methods

doi: 10.1016/j.jcyt.2012.07.001

Figure Lengend Snippet: Monitoring adipogenic progression of MSCs with the use of flow cytometry for FABP4 and Nile red. (A) Representative histogram plots for FABP4 staining. (B) The increase in SSC characteristics for the whole population of cells as the time course progresses; error bars represent standard deviation of the mean for three donors tested. (C) Representative histogram plots for Nile red staining. (D) Nile red fluorescence of both populations of gated low- and high-SSC cells, demonstrating a parallel increase throughout the time course. Days 0 and 3 are omitted because of very low frequency of high-SSC early in differentiation. Numbers in the top right corners of histograms represent median fluorescence intensities. FI is fluorescent intensity.

Article Snippet: FABP4 protein was detected with the use of a purified polyclonal goat anti-human FABP4 antibody (IgG, R & D Systems, Abingdon, UK) at 1:20 dilution in PBS.

Techniques: Flow Cytometry, Staining, Standard Deviation, Fluorescence

Journal: Cytotherapy

Article Title: Assay validation for the assessment of adipogenesis of multipotential stromal cells—a direct comparison of four different methods

doi: 10.1016/j.jcyt.2012.07.001

Figure Lengend Snippet: Advantages and disadvantages of different assays for the evaluation of MSC adipogenesis.

Article Snippet: FABP4 protein was detected with the use of a purified polyclonal goat anti-human FABP4 antibody (IgG, R & D Systems, Abingdon, UK) at 1:20 dilution in PBS.

Techniques: Staining, RNA Extraction, Quantitative Proteomics, Biomarker Discovery, TaqMan Assay, Flow Cytometry, Purification, Fluorescence, High Throughput Screening Assay, Microscopy

Journal: Bone Research

Article Title: FABP4 secreted by M1-polarized macrophages promotes synovitis and angiogenesis to exacerbate rheumatoid arthritis

doi: 10.1038/s41413-022-00211-2

Figure Lengend Snippet: The expression of FABP4 in the RA mouse model, BMDMs and RA patients. a , b FABP4 concentrations in the synovial fluid ( a ) and serum ( b ) of controls and RA patients ( n = 8 per group) were assessed by ELISA. C Representative images and quantification of HE staining and FABP4 immunohistochemical staining in the synovium of control and C57BL/6 J mice at 4 and 8 weeks after AIA modeling ( n = 10 per group). Scale bars: 50 μm, 100 μm and 200 μm. d Representative images and quantitative analysis of coimmunostaining of FABP4 with F4/80 or NOS2 in the synovium of control and C57BL/6 J mice at 4 and 8 weeks after AIA modeling ( n = 10 per group). Scale bars: 12.5 μm. e Representative images and quantification of safranin O and fast green staining and FABP4 immunohistochemical staining in knee cartilage from controls and C57BL/6 J mice at 4 and 8 weeks after AIA modeling ( n = 10 per group). Scale bars: 50 μm and 100 μm. f Western blot showing FABP4 and NOS2 in BMDMs stimulated with 50, 200, or 500 ng·mL −1 LPS for 12 h or 24 h. g Quantitative PCR analysis of NOS2, Arg-1, and FABP4 mRNA expression in BMDMs ( n = 3 per group). h FABP4 concentrations in the supernatant of BMDMs stimulated with 50, 200, or 500 ng·mL −1 LPS for 12 h or 24 h ( n = 3 per group) were assessed by ELISA. Student’s t -test or one-way analysis of variance (ANOVA) and Tukey’s multiple comparison test. * P < 0.05, ** P < 0.01; ns, no significance. The data are shown as the mean ± SEM

Article Snippet: We used human and mouse FABP4 ELISA kits (Elabscience Biotechnology, Bethesda, MD, USA: #E-EL-H0285c and #E-EL-M2404c) to analyze the level of FABP4 in the supernatant of macrophages stimulated with lipopolysaccharide (LPS), the serum of C57BL/6 J and TSC1KO mice, and human serum and synovial fluid.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Staining, Immunohistochemical staining, Control, Western Blot, Real-time Polymerase Chain Reaction, Comparison

Journal: Bone Research

Article Title: FABP4 secreted by M1-polarized macrophages promotes synovitis and angiogenesis to exacerbate rheumatoid arthritis

doi: 10.1038/s41413-022-00211-2

Figure Lengend Snippet: The effect of FABP4 and M1-polarized macrophage supernatant on HUVECs, FLSs, and chondrocytes in vitro. a , b Tube formation assay and quantification of HUVECs cultured with vehicle (C), rhFABP4 (P), rhFABP4 + BMS309403 (PB), M1-polarized macrophage supernatant (S), or M1-polarized macrophage supernatant+BMS309403 (SB) ( n = 3 per group). Scale bar: 100 µm. c Immunoblot analysis of FABP4 and VEGFα in HUVECs cultured with C, P, PB, S, or SB for 24 h. d , g Representative images and quantification of BrdU (green) immunofluorescence ( d ) and Transwell assays ( g ) in HUVECs and FLSs treated with C, P, PB, S, or SB for 24 h ( n = 3 per group). Scale bar: 25 µm, 100 µm. e , f Immunoblot analysis of ERK1/2, p-ERK1/2, P65, and p-P65 in HUVECs ( e ) and FLSs ( f ) treated with C, P, PB, S, or SB for 1 h. h Toluidine blue staining of ATDC5 cells treated with C, P, PB, S, or SB for 24 h ( n = 3 per group). i Immunoblot analysis of FABP4, MMP13, Sox9, and Col2a1 in primary chondrocytes treated with C, P, PB, S, or SB for 24 h. j Immunoblot analysis of P65 and p-P65 in primary chondrocytes treated with C, P, PB, S, or SB for 1 h. One-way ANOVA and Tukey’s multiple comparison test. * P < 0.05, ** P < 0.01; # P < 0.05, ## P < 0.01 compared to the control. The data are shown as the mean ± SEM

Article Snippet: We used human and mouse FABP4 ELISA kits (Elabscience Biotechnology, Bethesda, MD, USA: #E-EL-H0285c and #E-EL-M2404c) to analyze the level of FABP4 in the supernatant of macrophages stimulated with lipopolysaccharide (LPS), the serum of C57BL/6 J and TSC1KO mice, and human serum and synovial fluid.

Techniques: In Vitro, Tube Formation Assay, Cell Culture, Western Blot, Immunofluorescence, Staining, Comparison, Control

Journal: Bone Research

Article Title: FABP4 secreted by M1-polarized macrophages promotes synovitis and angiogenesis to exacerbate rheumatoid arthritis

doi: 10.1038/s41413-022-00211-2

Figure Lengend Snippet: Recombinant FABP4 exacerbates the development of RA in C57BL/6 J mice. a Representative images and quantitative analysis of FABP4 were assessed by immunohistochemical staining and coimmunostaining of FABP4 with F4/80 or NOS2 in the synovium of control and RA mice treated with vehicle or rmFABP4 for 4 and 8 weeks ( n = 10 per group). Scale bars: 12.5 µm and 100 µm. b – i Representative images and quantification of Vimentin and MMP3 coimmunostaining ( b , f ), CD31 and EMCN coimmunostaining ( c , g ), Col2a1 immunofluorescence staining ( d , h ), and MMP13 immunohistochemical staining ( e , i ) in the knee joints of control and RA mice treated with vehicle or rmFABP4 for 4 and 8 weeks ( n = 10 per group). Scale bars: 12.5 µm, 25 µm, and 100 µm. One-way ANOVA and Tukey’s multiple comparison test. ** P < 0.01. The data are shown as the mean ± SEM

Article Snippet: We used human and mouse FABP4 ELISA kits (Elabscience Biotechnology, Bethesda, MD, USA: #E-EL-H0285c and #E-EL-M2404c) to analyze the level of FABP4 in the supernatant of macrophages stimulated with lipopolysaccharide (LPS), the serum of C57BL/6 J and TSC1KO mice, and human serum and synovial fluid.

Techniques: Recombinant, Immunohistochemical staining, Staining, Control, Immunofluorescence, Comparison

Journal: Bone Research

Article Title: FABP4 secreted by M1-polarized macrophages promotes synovitis and angiogenesis to exacerbate rheumatoid arthritis

doi: 10.1038/s41413-022-00211-2

Figure Lengend Snippet: Activation of the mTORC1 pathway enhances the secretion of FABP4 by M1-polarized macrophages to exacerbate RA progression. a , b Representative images and quantification of F4/80 and pS6 coimmunostaining in the mouse ( n = 10 per group) ( a ) and human synovium ( n = 16 per group) ( b ) of the control and RA groups. Scale bar: 12.5 µm. c Western blot showing S6 and pS6 in human synovial tissue. d Western blot showing FABP4, NOS2, S6 and pS6 in BMDMs treated with LPS or rapamycin. e Representative immunofluorescence staining images and quantitative analysis of FABP4 in BMDMs stimulated with LPS or rapamycin ( n = 3 per group). Scale bar: 100 µm. f Western blot showing FABP4, NOS2, S6 and pS6 in BMDMs treated with MHY1485 or rapamycin. g Representative immunofluorescence staining images and quantitative analysis of FABP4 in BMDMs treated with MHY1485 or rapamycin ( n = 3 per group). Scale bar: 100 µm. h Western blot showing FABP4, NOS2, S6 and pS6 in BMDMs from control and TSC1KO mice. i Representative images and quantification of F4/80 and pS6 coimmunostaining in the synovium of wild-type and TSC1KO mice at 4 and 8 weeks after AIA modeling ( n = 10 per group). Scale bar: 12.5 µm. j Representative images and quantification of FABP4 immunohistochemical staining and coimmunostaining of FABP4 with F4/80 or NOS2 in the synovium of control and TSC1KO mice at 4 and 8 weeks after AIA modeling ( n = 10 per group). Scale bars: 12.5 µm and 100 µm. f–j Representative images ( f ) and quantification of Vimentin and MMP3 coimmunostaining ( g ), CD31 and EMCN coimmunostaining ( h ), Col2a1 immunofluorescence staining ( i ), and MMP13 immunohistochemical staining ( j ) in the knee joints of controls and TSC1KO mice at 4 and 8 weeks after AIA modeling ( n = 10 per group). Scale bar: 12.5 µm, 25 µm, and 100 µm. Student’s t -test or one-way ANOVA and Tukey’s multiple comparison test. ** P < 0.01. The data are shown as the mean ± SEM

Article Snippet: We used human and mouse FABP4 ELISA kits (Elabscience Biotechnology, Bethesda, MD, USA: #E-EL-H0285c and #E-EL-M2404c) to analyze the level of FABP4 in the supernatant of macrophages stimulated with lipopolysaccharide (LPS), the serum of C57BL/6 J and TSC1KO mice, and human serum and synovial fluid.

Techniques: Activation Assay, Control, Western Blot, Immunofluorescence, Staining, Immunohistochemical staining, Comparison

Journal: Bone Research

Article Title: FABP4 secreted by M1-polarized macrophages promotes synovitis and angiogenesis to exacerbate rheumatoid arthritis

doi: 10.1038/s41413-022-00211-2

Figure Lengend Snippet: Inhibition of the mTORC1 pathway reduces FABP4 secretion by M1-polarized macrophages to attenuate RA progression. a Representative images and quantification of F4/80 and pS6 coimmunostaining in the synovium of controls and Rheb1KO mice at 4 and 8 weeks after AIA modeling ( n = 10 per group). Scale bar: 12.5 µm. b – e Representative images ( b ) and quantitative analysis of FABP4 immunohistochemical staining ( c ) and coimmunostaining of FABP4 with F4/80 ( d ) or NOS2 ( e ) in the synovium of controls and Rheb1KO mice at 4 and 8 weeks after AIA modeling ( n = 10 per group). Scale bars: 12.5 µm and 100 µm. f – j Representative images ( f ) and quantitative analysis of Vimentin and MMP3 coimmunostaining ( g ), CD31 and EMCN coimmunostaining ( h ), Col2a1 immunofluorescence staining ( i ), and MMP13 immunohistochemical staining ( j ) in the knee joints of controls and Rheb1KO mice at 4 and 8 weeks after AIA modeling ( n = 10 per group). Scale bar: 12.5 µm, 25 µm, and 100 µm. Student’s t -test. ** P < 0.01. The data are shown as the mean ± SEM

Article Snippet: We used human and mouse FABP4 ELISA kits (Elabscience Biotechnology, Bethesda, MD, USA: #E-EL-H0285c and #E-EL-M2404c) to analyze the level of FABP4 in the supernatant of macrophages stimulated with lipopolysaccharide (LPS), the serum of C57BL/6 J and TSC1KO mice, and human serum and synovial fluid.

Techniques: Inhibition, Immunohistochemical staining, Staining, Immunofluorescence

Journal: Bone Research

Article Title: FABP4 secreted by M1-polarized macrophages promotes synovitis and angiogenesis to exacerbate rheumatoid arthritis

doi: 10.1038/s41413-022-00211-2

Figure Lengend Snippet: BMS309403 and anagliptin reduce FABP4 expression in murine synovial macrophages. a – c Representative images and quantification of FABP4 immunohistochemical staining and coimmunostaining of FABP4 with F4/80 or NOS2 in the synovium of TSC1KO mice treated with vehicle, BMS309403 or anagliptin for 4 and 8 weeks after AIA modeling ( n = 10 per group). Scale bar: 12.5 µm and 100 µm. d – f Representative images and quantification of FABP4 immunohistochemical staining and coimmunostaining of FABP4 and F4/80 or NOS2 in the synovium of RA mice treated with vehicle or BMS309403 for 4 and 8 weeks ( n = 10 per group). Scale bar: 12.5 µm and 100 µm. Student’s t -test or one-way ANOVA and Tukey’s multiple comparison test. * P < 0.05, ** P < 0.01. The data are shown as the mean ± SEM

Article Snippet: We used human and mouse FABP4 ELISA kits (Elabscience Biotechnology, Bethesda, MD, USA: #E-EL-H0285c and #E-EL-M2404c) to analyze the level of FABP4 in the supernatant of macrophages stimulated with lipopolysaccharide (LPS), the serum of C57BL/6 J and TSC1KO mice, and human serum and synovial fluid.

Techniques: Expressing, Immunohistochemical staining, Staining, Comparison

Journal: Bone Research

Article Title: FABP4 secreted by M1-polarized macrophages promotes synovitis and angiogenesis to exacerbate rheumatoid arthritis

doi: 10.1038/s41413-022-00211-2

Figure Lengend Snippet: Inhibiting FABP4 prevents RA development. a – d Representative images of Vimentin and MMP3 coimmunostaining ( a ), CD31 and EMCN coimmunostaining ( b ), Col2a1 immunofluorescence staining ( c ), and immunohistochemical staining of MMP13 ( D ) in the knee joints of TSC1KO mice treated with vehicle, BMS309403 or anagliptin for 4 and 8 weeks after AIA modeling. Scale bar: 12.5 µm, 25 µm, and 100 µm. e – h Representative images of Vimentin and MMP3 coimmunostaining ( e ), CD31 and EMCN coimmunostaining ( f ), Col2a1 immunofluorescence staining ( g ), and immunohistochemical staining of MMP13 ( h ) in the knee joints of RA mice treated with vehicle or BMS309403 for 4 and 8 weeks. Scale bar: 12.5 µm, 25 µm, and 100 µm. i – l Quantification of Vimentin-MMP3 ( i ), CD31-EMCN ( j ), Col2a1 ( k ), and MMP13 ( l ) in TSC1KO mice treated with vehicle, BMS309403, or anagliptin for 4 and 8 weeks after AIA surgery ( n = 10 per group). m – p Quantification of Vimentin-MMP3 ( m ), CD31-EMCN ( n ), Col2a1 ( o ), and MMP13 ( p ) in RA mice treated with vehicle or BMS309403 for 4 and 8 weeks ( n = 10 per group). Student’s t -test or one-way ANOVA and Tukey’s multiple comparison test were used. ** P < 0.01. The data are shown as the mean ± SEM

Article Snippet: We used human and mouse FABP4 ELISA kits (Elabscience Biotechnology, Bethesda, MD, USA: #E-EL-H0285c and #E-EL-M2404c) to analyze the level of FABP4 in the supernatant of macrophages stimulated with lipopolysaccharide (LPS), the serum of C57BL/6 J and TSC1KO mice, and human serum and synovial fluid.

Techniques: Immunofluorescence, Staining, Immunohistochemical staining, Comparison