Journal: Arthritis Research & Therapy

Article Title: Connective tissue growth factor promotes articular damage by increased osteoclastogenesis in patients with rheumatoid arthritis

doi: 10.1186/ar2863

Figure Lengend Snippet: Connective tissue growth factor expression in synovial tissue of patients with rheumatoid arthritis. The representative results of HE staining (Figure 2A), immunofluorescence anti-CTGF antibody staining (Figure 2B; green), and anti-F4/80 antibody staining (Figure 2C; red) are shown using surgical samples from RA and OA patients. The samples were counterstained by DAPI (blue) for nuclear staining and merge images are shown (Figure 2D). A strong expression of CTGF and F4/80 was observed in the samples of RA compared to OA and the CTGF expression cells were not overlapped with F/40 expression cells indicating that CTGF is upregulated in synovial fibroblasts rather than macrophages.

Article Snippet: The serum level of CTGF in human sera was evaluated by a sandwich ELISA system using two different anti-human CTGF antibodies; monoclonal anti-human CTGF antibody (R&D System, Cat#MAB660) and biotinated anti-human CTGF antibody (R&D System, Cat#BAF660).

Techniques: Expressing, Staining, Immunofluorescence

Journal: Arthritis Research & Therapy

Article Title: Connective tissue growth factor promotes articular damage by increased osteoclastogenesis in patients with rheumatoid arthritis

doi: 10.1186/ar2863

Figure Lengend Snippet: Synergistic effects of connective tissue growth factor on M-CSF/sRANKL-mediated osteoclastic function. Figure 5A shows the results of the resorption of osteoclasts on calcium phosphate. Vacant regions indicated by arrows represent the areas where the osteoclasts actually absorbed. There was no vacant region in negative control cells (M-CSF alone). In contrast to negative control, significant vacant regions were observed in osteoclasts induced by M-CSF/sRANKL. CTGF further expanded the vacant areas in combination with M-CSF/sRANKL and anti-CTGF antibody neutralized this effect. Figure 5B shows the levels of expression of osteoclasts specific proteases (MMP-9 and cathepsin-K) measured by quantitative real time RT-PCR. Synergistic effect of CTGF was also observed for M-CSF/sRANKL-mediated osteoclastogenesis. Bars in Figure 5B indicate the SD.

Article Snippet: The serum level of CTGF in human sera was evaluated by a sandwich ELISA system using two different anti-human CTGF antibodies; monoclonal anti-human CTGF antibody (R&D System, Cat#MAB660) and biotinated anti-human CTGF antibody (R&D System, Cat#BAF660).

Techniques: Negative Control, Expressing, Quantitative RT-PCR

Journal: Arthritis Research & Therapy

Article Title: Connective tissue growth factor promotes articular damage by increased osteoclastogenesis in patients with rheumatoid arthritis

doi: 10.1186/ar2863

Figure Lengend Snippet: Connective tissue growth factor mediate ERK1/2 and focal adhesion kinase activation through integrin αVβ3 signal transduction. Figure 6A shows the immnoprecipitation and immunoblotting analysis. The cell extracts of osteoclasts stimulated with recombinant CTGF (10 or 50 ng/ml) at 60 minutes were precipitated using anti-integrin αVβ3 antibody and subsequently blotted with anti- phosphorylated ERK1/ERK2, conventional ERK1/ERK2, and integrin αVβ3 antibodies respectively. The phosphorylated ERK1/ERK2 was recruited with integrin αVβ3 by CTGF stimulation. Figure 6B shows the immunoblotting analysis using anti- phosphorylated FAK, conventional FAK, and β-actin antibodies in the osteoclasts extracts treated with CTGF (10 ng/ml) at 5, 15, 60, and 120 minutes in the presence or absence of anti-CTGF antibody (1 μg/ml). CTGF stimulation resulted in phosphorylation of FAK from 60 minutes and this effect was neutralized by anti-CTGF antibody suggesting activation of signal transduction pathways through integrin αVβ3.

Article Snippet: The serum level of CTGF in human sera was evaluated by a sandwich ELISA system using two different anti-human CTGF antibodies; monoclonal anti-human CTGF antibody (R&D System, Cat#MAB660) and biotinated anti-human CTGF antibody (R&D System, Cat#BAF660).

Techniques: Activation Assay, Transduction, Western Blot, Recombinant, Phospho-proteomics

Journal: bioRxiv

Article Title: CD36-Pyruvate Kinase M2 Signaling Promotes Macrophage Phagocytosis Through Mitochondrial Reactive Oxygen Species

doi: 10.1101/2023.09.07.556574

Figure Lengend Snippet: A , a diagram of the ex vivo mtROS and phagocytosis assay. B , Total amount of aortic F4/80 + /Trem2 + macrophages were quantified and shown in the bar graph; n=4-7 individual mice per group. C , MitoNeoD MFI was quantified in aortic F4/80 + /Trem2 + macrophages and shown in the bar graph; n=5 individual mice per group. D , pHrodo MFI was quantified in aortic F4/80 + /Trem2 + macrophages and shown in the bar graph; n=5-6 individual mice per group. E , a diagram of the in vivo phagocytosis assay. F , pHrodo MFI was quantified in aortic F4/80 + /Trem2 + macrophages and shown in the bar graph; n=5-7 individual mice per group.

Article Snippet: For phagocytosis assays, the filtered single-cell suspension was incubated with 20 μg/ml pHrodo-conjugated E. coli bioparticles in RPMI1640 with 10% FBS at 37°C for 30 min, followed by immunostaining with PE/Cy5-conjugated F4/80 antibody (BioLegend, Cat#123111) and PE-conjugated Trem2 antibody (R&D Systems, Cat#FAB17291P) in Flow Buffer at RT for 15 min in the dark.

Techniques: Ex Vivo, Phagocytosis Assay, In Vivo

Journal: bioRxiv

Article Title: CD36-Pyruvate Kinase M2 Signaling Promotes Macrophage Phagocytosis Through Mitochondrial Reactive Oxygen Species

doi: 10.1101/2023.09.07.556574

Figure Lengend Snippet: A-D , Mouse scRNA-seq data were re-analyzed from a previous publication . Uniform manifold approximation and projection (UMAP) representation of 11 aortic CD45 + immune cell clusters were shown in A . Trem2 gene expression pattern ( B ) and Pkm gene expression pattern ( C ) were shown in the UMAP. D , Violin plots show the Pkm and Trem2 expression distribution among aortic macrophage subpopulations. ResMac: resident macrophages; InflaMac: inflammatory macrophages. E , HMDMs transfected with PKM siRNA were treated with 50 μg/ml oxLDL for 24 h before subjected to mtROS assay (left panel) or phagocytosis assay (right panel). MFI was quantified and shown in the bar graph; n=4-5 per group. F , Representative confocal images of macrophages immunostained for PKM2 (green) and Tom20 (red). Nuclei were stained by DAPI (blue). Scale bar: 5 μm. G , HMDMs treated with 20 μg/ml LDL (control) or oxLDL for 3 h were lysed, subjected to cell fractionation into mitochondrial and cytosol fractions. PKM2 and ATP5A (mitochondria fraction loading control) blot images from mitochondrial fractions were shown on the left. PKM2 and β-actin (cytosol fraction loading control) blot images from cytosol fractions were shown on the right. Images were quantified, normalized to each loading control, and expressed as fold change of control. n=4 per group. H , WT or Cd36 -null peritoneal macrophages treated with 20 μg/ml LDL (control) or oxLDL for 3 h and then processed as in G. Mitochondrial fractions were immunoblotted for PKM2 and ATP5A and blot images were shown. Images were quantified, normalized, and expressed as fold change of control. n=4 per group. I , WT macrophages treated with 20 μg/ml oxLDL or pre-treated 1 or 5μM shikonin before addition of oxLDL, incubating for 3 h, and then processed as in G. Mitochondrial fractions were immunoblotted for PKM2 and Tom20 and blot images were shown. Images were quantified and expressed as fold change of control. n=3 per group. J , WT macrophages pre-treated with 20 μg/ml oxLDL or in combination with 1 μM shikonin for 24 h before mtROS or phagocytosis assay. The MitoNeoD (left) or pHrodo (right) MFI was quantified and shown in the bar graph; n=3-4 per group.

Article Snippet: For phagocytosis assays, the filtered single-cell suspension was incubated with 20 μg/ml pHrodo-conjugated E. coli bioparticles in RPMI1640 with 10% FBS at 37°C for 30 min, followed by immunostaining with PE/Cy5-conjugated F4/80 antibody (BioLegend, Cat#123111) and PE-conjugated Trem2 antibody (R&D Systems, Cat#FAB17291P) in Flow Buffer at RT for 15 min in the dark.

Techniques: Gene Expression, Expressing, Transfection, Phagocytosis Assay, Staining, Control, Cell Fractionation

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Temporal Proteomic and Phosphoproteomic Profiling Deciphers Molecular Dynamics of Acute-to-Chronic Kidney Disease After Ischemia-Reperfusion Injury, With Dock2 Emerging as a Key Regulator

doi: 10.1016/j.mcpro.2026.101509

Figure Lengend Snippet: Ischemia-reperfusion injury elicits time-dependent changes of TECs injury, inflammatory infiltration, and interstitial fibrotic remodeling. A , schematic illustration of the experimental protocol for inducing renal IRI in a mouse model. B , levels of Scr from sham-operated mice and mice sacrificed at 1 h, 1 day, 3 days, 7 days, 28 days after I/R. C and D , protein expression profiling by western blot ( C ) and subsequent statistical analysis ( D ) of Havcr1/Kim1 in renal cortical samples. E , immunohistochemical detection of Havcr1/Kim1 expression in renal cortical sections from sham and I/R-treated mice. The scale bar represents 100 μm. F and G , protein expression profiling by western blot ( F ) and subsequent statistical analysis ( G ) of IL-6 in renal cortical samples. H , immunofluorescent staining for the macrophage marker F4/80 in renal cortical sections. The scale bar represents 40 μm. I , histopathological assessment of renal tissue architecture (H&E) and collagen deposition (Masson’s trichrome) in experimental groups. The scale bar represents 100 μm. Data are presented as mean ± SEM. n.s, not significant; ∗ p < 0.05; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. Havcr, hepatitis A virus cellular receptor 1; IL-6, interleukin-6; IRI, ischemia-reperfusion injury; Kim1, kidney injury molecule-1; Scr, serum creatinine; TEC, tubular epithelial cell.

Article Snippet: Following blocking, the sections were incubated overnight at 4 °C with an anti-Havcr1/Kim1 primary antibody (R&D Systems, cat. AF1817, dilution 1:200), then treated with horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature (RT) for 1 h, and finally developed with 3,3′-diaminobenzidine (DAB) substrate (ZSGB, ZLI-9017).

Techniques: Expressing, Western Blot, Immunohistochemical staining, Staining, Marker, Virus

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Temporal Proteomic and Phosphoproteomic Profiling Deciphers Molecular Dynamics of Acute-to-Chronic Kidney Disease After Ischemia-Reperfusion Injury, With Dock2 Emerging as a Key Regulator

doi: 10.1016/j.mcpro.2026.101509

Figure Lengend Snippet: Dock family proteins are identified, with knockdown of Dock2 demonstrating a significant attenuation of the proinflammatory response of TECs in vitro . A , heatmap of Dock family proteins and phosphosites (ANOVA, p < 0.05). B , intensity plots showing Dock2 ( black line ) and its phosphosite S1704 (Dock2_pS1704, green line ) across the six experimental groups. C and D , protein expression profiling by western blot ( C ) and subsequent statistical analysis ( D ) of Dock2 in renal cortical samples. E , immunofluorescent detection of Dock2 and Havcr1/Kim1 expression and colocalization in renal tissue sections from sham and I/R-treated mice. The scale bar represents 40 μm. F , the knockdown efficiency of Dock2 by siRNA in H/R-treated HK-2 cells was analyzed by western blot. G , the statistical analysis of ( F ). H – J , RT-qPCR showing the effects of Dock2 knockdown on mRNA expression levels of MCP-1, TNF-α, and IL-6. K , western blot analysis was performed to assess the effects of Dock2 knockdown on IKK-β phosphorylation in H/R-treated HK-2 cells. L , densitometric analysis of p-IKKβ from ( K ) was performed with normalization to the respective total protein. M , translocation of NF-κB p65 in HK-2 cells was detected by immunofluorescence. NF-κB p65 ( green ), DAPI ( blue ). The scale bar represents 5 μm. Data are presented as mean ± SEM. n.s, not significant; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. DAPI, 4′,6-diamidino-2-phenylindole; Dock2, dedicator of cytokinesis 2; Havcr, hepatitis A virus cellular receptor 1; H/R, hypoxia/reoxygenation; IKKβ, IκB kinase beta; Kim1, kidney injury molecule-1; IL-6, interleukin-6; MCP-1, monocyte chemoattractant protein-1; RT-qPCR, reverse transcription quantitative PCR; TEC, tubular epithelial cell; TNF-α, tumor necrosis factor-α.

Article Snippet: Following blocking, the sections were incubated overnight at 4 °C with an anti-Havcr1/Kim1 primary antibody (R&D Systems, cat. AF1817, dilution 1:200), then treated with horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature (RT) for 1 h, and finally developed with 3,3′-diaminobenzidine (DAB) substrate (ZSGB, ZLI-9017).

Techniques: Knockdown, In Vitro, Phospho-proteomics, Expressing, Western Blot, Quantitative RT-PCR, Translocation Assay, Immunofluorescence, Virus, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Temporal Proteomic and Phosphoproteomic Profiling Deciphers Molecular Dynamics of Acute-to-Chronic Kidney Disease After Ischemia-Reperfusion Injury, With Dock2 Emerging as a Key Regulator

doi: 10.1016/j.mcpro.2026.101509

Figure Lengend Snippet: Inhibition of Dock2 by CPYPP attenuates tubular injury, inflammatory infiltration and interstitial fibrosis after renal IRI in mice. A , schematic of the experimental design for unilateral I/R in mice treated with CPYPP or vehicle via i.p. injection following I/R induction. B , measurement of Scr levels. C , H&E staining was performed to assess the effect of pharmacological inhibition of Dock2 by CPYPP on renal tissue architecture in mice at 3 days and 28 days post-I/R. Inflammatory cells, tubular casts, and dilated tubules are indicated by solid arrows , hollow arrows , and triangles , respectively. The scale bar represents 100 μm. D , tubular injury score at 3 days post-I/R (quantified from C ). E – I , western blot ( E ) and quantitative analysis ( F – I ) of renal cortical Havcr1/Kim1, IL-6, TNF-α, and MCP-1 expression in sham-operated and 3 days post-I/R mice, with or without CPYPP treatment. J , immunofluorescent staining for the macrophage marker F4/80 in renal cortical sections to assess inflammatory infiltration. The scale bar represents 40 μm. K , the quantitative analysis of the F4/80 immunofluorescence staining from ( J ). L – N , western blot ( L ) and quantitative analysis ( M and N ) of renal cortical FN and α-SMA expression in sham-operated and 28 days post-I/R mice, with or without CPYPP treatment. O , Masson's trichrome staining was used to evaluate the effect of CPYPP on renal collagen deposition at 28 days post-I/R, with quantification data shown in ( P ). The scale bar represents 100 μm. Data are presented as mean ± SEM. n.s, not significant; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. CPYPP, 4-[3′-(2″-chlorophenyl)-2′-propen-1′-ylidene]-1-phenyl-3,5-pyrazolidinedione; Dock2, dedicator of cytokinesis 2; FN, fibronectin; Havcr, hepatitis A virus cellular receptor 1; IL-6, interleukin-6; IRI, ischemia-reperfusion injury; Kim1, kidney injury molecule-1; MCP-1, monocyte chemoattractant protein-1; α-SMA, alpha-smooth muscle actin; Scr, serum creatinine; TNF-α, tumor necrosis factor-α.

Article Snippet: Following blocking, the sections were incubated overnight at 4 °C with an anti-Havcr1/Kim1 primary antibody (R&D Systems, cat. AF1817, dilution 1:200), then treated with horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature (RT) for 1 h, and finally developed with 3,3′-diaminobenzidine (DAB) substrate (ZSGB, ZLI-9017).

Techniques: Inhibition, Injection, Staining, Western Blot, Expressing, Marker, Immunofluorescence, Virus