Journal: Cellular and Molecular Life Sciences

Article Title: SEMA6C: a novel adhesion-independent FAK and YAP activator, required for cancer cell viability and growth

doi: 10.1007/s00018-023-04756-1

Figure Lengend Snippet: Sema6C-induced signaling cascade in cancer cells, mediated by c-Abl and FAK kinases. A Western blotting analysis and densitometric values of phosphorylated FAK protein in multiple cancer cells subjected to Sema6C knock-down, Sema6C-overexpression, or controls; images show representative results of consistent replicate experiments. Total FAK levels provided a protein loading control and were used in the normalization of band intensity analyses; the indicated values (N = 3 replicates/group) represent fold-changes in Sema6C-modulated cells vs. each respective control. B Western blotting analysis of phospho-FAK, total FAK, and YAP levels in detached Sema6C-overexpressing and control BxPC-3 cells in suspension; GAPDH provided a protein loading control. The image shows representative results consistently reproduced in two independent experiments, summarized in the graph on the right showing average values ± SD; statistical analysis was done by unpaired Student’s t test: **p < 0.01; ***p < 0.001. C Immunofluorescence confocal analysis of E-cadherin (red) and YAP subcellular localization (green) in Sema6C-overexpressing BxPC-3 cells treated with the FAK-inhibitor PF-573228 at concentrations 0.5 μM (for 16 h) or 5 μM (1 h), or subjected to FAK knock-down by shRNA transfection (see Suppl. Fig. 12C, D). Scale bars: 25 μm. The graph on the right indicates the cell fraction containing nuclear localized YAP (average values ± SD) (N = 3). Statistical analysis was done by two-way ANOVA: ****p < 0.0001. D Western blotting analysis and densitometric values of phosphorylated c-Abl protein in cancer cells bearing Sema6C-overexpression, or controls; images are representative of at least two independent replicas yielding consistent results. Total Abl levels provided a loading control and were used in the normalization of band intensity analyses; the indicated values (N = 3 replicates/group) represent fold-changes in Sema6C-OE cells vs. respective controls. E Co-immunoprecipitation experiments performed by incubating protein lysates of Sema6C-OE or mock control HCT116 cells with anti-Abl coated beads (or beads coated with a matched unrelated antibody, anti-ID3); the separated immunocomplexes were then probed to detect the presence of c-Abl kinase and any associated Sema6C protein. Detection of antibody heavy chains (IgG-HC) provided a control of immunocomplex loading. Data are representative of four independent experiments with consistent results. Similar results were obtained in BxPC3 cells, shown in Suppl. Fig. 15A. F Western blotting analysis of phosphorylated FAK and ERK proteins in Sema6C-OE BxPC-3 cells incubated (or not) with 10 μM imatinib for 2 h, and in mock cells for comparison. GAPDH levels provided a common loading control and were used in the normalization of band intensity analyses. Data shown are representative of two independent experiments with consistent results

Article Snippet: Moreover, PF-573228 (Tocris Bioscience Cat. #3239), Verteporfin (Sigma-Aldrich; Cat. #SML0534), Imatinib (Cell signaling, Cat. #9084).

Techniques: Western Blot, Knockdown, Over Expression, Control, Suspension, Immunofluorescence, shRNA, Transfection, Immunoprecipitation, Incubation, Comparison

Journal: bioRxiv

Article Title: GABA-independent activation of GABAB receptor by mechanical forces

doi: 10.1101/2025.06.09.658551

Figure Lengend Snippet: a , Schematic illustration depicting the modulation of GABA B receptor activity by ligands (e.g. agonists: GABA and baclofen; antagonist: CGP54626). b, Schematic representation of the experiments applying traction force and shear stress to cells in this study, encompassing conditions such as cell suspension or adhesion; PDL or FN coating; and shear stress treatment. No GABA was added in these experiments. c, IP 1 production in HEK293 cells transfected with vector or GABA B receptor, along with G qi9 chimera in the corresponding conditions: suspension or adhesion (left) ; PDL or FN coating (middle) , without (control) or with shear stress (15 dyn/cm , 15 min) (right) . Data are present as mean ± s.e.m. from five biologically independent experiments each performed in triplicates and analyzed using unpaired t test (two-tailed) to determine significance. *** P < 0.001, * P < 0.05, not significant (ns) > 0.05. d, G i protein activation in HEK293 cells transfected with vector or GABA B receptor, along with G i protein sensors under suspension, PDL-coating or FN-coating condition. Left: Schematic presentation of G i protein activity measurement using G i protein BRET sensor. Right: BRET ratio of cells in suspension or cells cultured in PDL-coating or FN-coating dishes. Data are present as mean ± s.e.m. from nine biologically independent experiments for vector and GABA B receptor respectively each performed in triplicates, and analyzed using repeat measurements one-way ANOVA with Tukey’s multiple comparisons test to determine significance. *** P < 0.001, ** P < 0.01, not significant (ns) > 0.05. e, Real-time recording of intracellular Ca 2+ release in HEK293 cells expressing GABA B receptor and G qi9 . Left: Schematic presentation of shear stress loading device and real-time recording of Ca 2+ response in single cells. Right: Real-time Ca 2+ signal measurement. After the recording of basal state of Ca 2+ release for 50 seconds, cells were subjected to shear stress for 100 seconds. Shear stress was then halted for 150 seconds, after which baclofen was injected for 200 seconds. Data are present as mean ± s.e.m. from 85 cells recorded.

Article Snippet: CGP54626 hydrochloride (#HY-101378) and RGDS peptide (Arg-Gly-Asp-Ser, #HY-12290) were from Med Chem Express (Shanghai, China).

Techniques: Activity Assay, Shear, Suspension, Transfection, Plasmid Preparation, Control, Two Tailed Test, Activation Assay, Cell Culture, Expressing, Injection

Journal: bioRxiv

Article Title: GABA-independent activation of GABAB receptor by mechanical forces

doi: 10.1101/2025.06.09.658551

Figure Lengend Snippet: a , IP 1 production in HEK293 cells transfected with either control siRNA or integrin β3 siRNA, along with the expressing of vector and G qi9 , or GABA B receptor and G qi9 , under either adhesion or suspension conditions. Data are present as mean ± s.e.m. from five biologically independent experiments and analyzed using unpaired t test (two-tailed) to determine significance. *** P < 0.001, not significant (ns) > 0.05. b , Co-immunoprecipitation of GABA B receptor and integrin β 3 in HEK293 cells transfected with GABA B receptor constructs (Snap-tagged GB1 and GB2) using anti-integrin β 3 antibody, under basal condition. Only GB1 in cell surface was labeled and visualized using an impermeable Snap fluorescent substrate. c-f, Co-immunoprecipitation of GABA B receptor and integrin β 3 in HEK293 cells transfected with GABA B receptor constructs (HA-tagged GB1 and GB2) using anti-HA antibody, under conditions including suspension or adhesion (c) , without (control) or with shear stress (c) , Blebbistatin (50 μM, 30 min) treatment (d) , RGDS (10 μM, 12 h) treatment (e) , or CGP54626 (50 μM, 30 min) treatment (f) . Blots are representative from at least three biologically independent experiments ( c , suspension or adhesion, n = 3; control or with shear stress, n = 4; d , n = 3; e , n = 3; f , n = 4). The amount of integrin β 3 immunoprecipitated by IgG or HA antibody are present as mean ± s.e.m. in (e) and analyzed using unpaired t test (two-tailed) to determine significance. ** P < 0.01. g, Schematic representation of the BRET assay detecting direct interaction between GABA B receptor and integrin β 3 . Rluc was fused in C-terminal of GB1 or GB2 subunit (GB1 Rluc or GB2 Rluc ) as luminescence donor. Venus was fused in C-terminal of integrin β 3 or integrin α V subunit (integrin β 3Venus or integrin α Venus ) as fluorescence acceptor. h-i, Interaction of GABA B receptor and integrin β 3 interaction between GB1 and integrin β 3 or GB1 and integrin α V (h) , or GB2 and integrin β 3 (i) detected using BRET titration assay. Data were analyzed by nonlinear regression on a pooled data set from at least three biologically independent experiments each performed in triplicates, fitting with 1-site binding model in GraphPad Prism 8.

Article Snippet: CGP54626 hydrochloride (#HY-101378) and RGDS peptide (Arg-Gly-Asp-Ser, #HY-12290) were from Med Chem Express (Shanghai, China).

Techniques: Transfection, Control, Expressing, Plasmid Preparation, Suspension, Two Tailed Test, Immunoprecipitation, Construct, Labeling, Shear, Bioluminescence Resonance Energy Transfer, Fluorescence, Titration, Binding Assay

Journal: bioRxiv

Article Title: GABA-independent activation of GABAB receptor by mechanical forces

doi: 10.1101/2025.06.09.658551

Figure Lengend Snippet: a-d , IP 1 production in HEK293 cells transfected with the indicated constructs, along with G qi9 under either suspension or adhesion conditions: (a) : vector, GABA B receptor (GB1 and GB2), GB1 only or GB2 only; (b) : vector, GABA B receptor or GABA B -ΔG (mutant with L685 in GB2 substituted with Phenylalanine that fails to couple G protein); (c) : vector, GABA B receptor, or GABA B -ΔB (mutant with the residues S276 and E465 in GB1 mutated to Analine that fails to bind GABA); (d) : vector or GABA B receptor, treated with or without antagonist CGP54626 (50 μM, 30 min). Data are present as mean ± s.e.m. from at least three biologically independent experiments (n = 4, 6, 5, 5 for a-d respectively) each performed in triplicates, and analyzed using unpaired t test (two-tailed) to determine significance. *** P < 0.001, * P < 0.05, not significant (ns) > 0.05. e, Model of the traction force-induced GABA B receptor activation through the closure of GB1 VFT -induced LB2 GB1 and LB2 GB2 in contact. The traction force-activated GABA B receptor requires both GB1 and GB2, and relies on GB2 for G protein coupling. Whereas the traction force-activated GABA B receptor is independent of GABA binding, preventing VFT GB1 closure by antagonist CGP54626 GABA B receptor abolishs traction force-induced GABA B receptor activation, indicating the closure of GB1 VFT -induced LB2 GB1 and LB2 GB2 in contact is important for traction force-induced GABA B receptor activation.

Article Snippet: CGP54626 hydrochloride (#HY-101378) and RGDS peptide (Arg-Gly-Asp-Ser, #HY-12290) were from Med Chem Express (Shanghai, China).

Techniques: Transfection, Construct, Suspension, Plasmid Preparation, Mutagenesis, Two Tailed Test, Activation Assay, Binding Assay

Journal: bioRxiv

Article Title: GABA-independent activation of GABAB receptor by mechanical forces

doi: 10.1101/2025.06.09.658551

Figure Lengend Snippet: Antagonist CGP54626-bound GABA B receptor fails to interact with integrin β3 whereas integrin β3 interacts with GB1 VFT of GABA B receptor with constitutive activity. Mechanical force promotes GB1 VFT and integrin β3 interaction to stabilize the closure of GB1 VFT -induced LB2 GB1 and LB2 GB2 in contact, further inducing an allosteric re-arrangement of the GABA B receptor 7TM towards an TM6-TM6 active conformation, culminating in the asymmetric activation of the receptor with G protein under GB2. Mechanical force acts as a positive allosteric modulator to boost up GABA-induced GABA B receptor activation through interaction between GB1 VFT and integrin β3.

Article Snippet: CGP54626 hydrochloride (#HY-101378) and RGDS peptide (Arg-Gly-Asp-Ser, #HY-12290) were from Med Chem Express (Shanghai, China).

Techniques: Activity Assay, Activation Assay

Journal: Cellular & molecular immunology

Article Title: A SARS-CoV-2-specific CAR-T-cell model identifies felodipine, fasudil, imatinib, and caspofungin as potential treatments for lethal COVID-19.

doi: 10.1038/s41423-023-00985-3

Figure Lengend Snippet: Fig. 4 Felodipine, fasudil, imatinib, and caspofungin, screened by the SARS-CoV-2-S CAR-T-cell model, suppress cytokine release. A The flowchart for drug screening that can suppress SARS-CoV-2-induced inflammation based on the SARS-CoV-2-S CAR-T-cell model is shown. B S- 293T, SARS-CoV-2-S CAR-T, and THP1 cells (1:10:10) were coincubated and treated with or without individual drugs (10 μM) from an FDA- approved library (~1049) before measuring the secretion of IL8 in the culture supernatants by ELISA. C The culture supernatants from drugs with more than 70% inhibition of IL8 secretion screened from B were further subjected to measurement of IFNγ by ELISA. D T cells were treated with drugs with more than 70% inhibition of IFNγ secretion screened from C for three days followed by toxicity assay (mean ± s.e.m). E S-293T, SARS-CoV-2-S CAR-T, and THP1 cells (1:10:10) were coincubated and treated with or without felodipine (#1), fasudil (#2), imatinib (#3), or caspofungin (#4) (10 μM) for three days before measuring the secretion of cytokines by a multiplex bead array. The inhibition rate is presented by a heatmap. F SARS-CoV-2-S CAR-T cells coincubated with S-293T cells (5:1) were treated with or without felodipine (#1), fasudil (#2), imatinib (#3), or caspofungin (#4) (10 μM) for the indicated duration, followed by a cytotoxicity assay (mean ± s.e.m). G SARS-CoV-2-S CAR- T cells were incubated with control 293 T or S-293T cells at a ratio of 3:1 for two days, and cells in suspension were subjected to RNA-seq analysis. Genes up- and downregulated by S-293T cells are shown in a pie chart (FDR < 0.01, FC > 1.5). H SARS-CoV-2-S CAR-T cells incubated with S-293T cells were treated with or without felodipine, fasudil, imatinib, or caspofungin (10 μM) for two days, and cells in suspension were subjected to RNA-seq analysis. Genes that were upregulated by S-293T cells but suppressed by felodipine, fasudil, imatinib, and caspofungin are shown. I Hallmark gene set enrichment analysis for overlapping genes (n = 865) as described in H. J, K SARS-CoV-2-S CAR-T cells were incubated with S-293T cells and treated with or without felodipine, fasudil, imatinib, or caspofungin (10 μM) for two days. T cells in suspension were subjected to RT‒qPCR analysis (J) to examine the expression of genes as indicated or immunoblotting analysis (K) using antibodies as indicated (Student’s t test, unpaired, two-tailed, *P < 0.05; **P < 0.01; ***P < 0.001)

Article Snippet: For treatment, fasudil (HY-10341, MCE), caspofungin (HY-17006, MCE) or imatinib (HY50948, MCE) was dissolved in PBS, and felodipine predissolved in DMSO was then diluted with sesame oil (#S25527, Shanghai Yuanye).

Techniques: Drug discovery, Enzyme-linked Immunosorbent Assay, Inhibition, Multiplex Assay, Cytotoxicity Assay, Incubation, Control, Suspension, RNA Sequencing, Expressing, Western Blot, Two Tailed Test

Journal: Cellular & molecular immunology

Article Title: A SARS-CoV-2-specific CAR-T-cell model identifies felodipine, fasudil, imatinib, and caspofungin as potential treatments for lethal COVID-19.

doi: 10.1038/s41423-023-00985-3

Figure Lengend Snippet: Fig. 5 Felodipine, fasudil, imatinib, and caspofungin attenuate lethal inflammation, ameliorate severe pneumonia, and prevent mortality in SARS-CoV-2-infected Syrian hamsters. A Schematic representation of SARS-CoV-2 infection and drug treatment. Hamsters were intranasally inoculated with SARS-CoV-2 (1 × 104 PFU) and then treated with or without felodipine (i.p.), fasudil (i.p.), imatinib (i.g.), or caspofungin (i.p.) on the indicated days (n = 6). Hamsters intranasally inoculated with SARS-CoV-2 without treatment were used as controls. Body weight was measured daily. Animals were euthanized to collect tissue samples at Day 7 post infection for virological and histological analysis. B The body weight of hamsters in each group from Day 0 to 7 post infection is shown. Significance was calculated using two-way ANOVA and is shown in the table (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: nonsignificant). C The survival curve for hamsters in each group from Day 0 to 7 post infection is shown. Significance was calculated using the log-rank (Mantel‒Cox) test (**P < 0.01). D Gross images of lung tissues collected from hamsters at Day 7 post infection are shown. E Representative images after H&E staining for lung lobe sections in hamsters at Day 7 post infection are shown. Scale bars, 50 μm. F Comprehensive pathological scores based on the severity and percentage of injured areas for each lung lobe for lung sections shown in Fig. S3A are shown. Significance was calculated using one-way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: nonsignificant). G The expression of inflammatory genes in lung tissues collected from hamsters at Day 7 post infection was detected by RT‒qPCR and represented by a heatmap. Data presented are the normalized value to the mock group after normalization to the expression of β-actin. The significance test is shown in Fig. S3B (*P < 0.05; **P < 0.01; ***P < 0.001). H The levels of cytokines, including IL6, IL10, IFNγ, and IFNβ in the lung tissues collected from hamsters at Day 7 post infection were measured by ELISA. Significance was calculated using one-way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). I RNA was extracted from the lungs of hamsters as described in A at Day 7 post infection followed by RNA-seq analysis. The similarity in the gene expression profile for the mock and SARS-CoV-2-infected groups is shown by a PCA plot. J Genes regulated by SARS-CoV-2 infection are shown in a volcano plot. Blue and red dots represent down- and upregulated genes, respectively (FDR < 0.01, FC > 1.5). K Genes that were upregulated by SARS-CoV-2 infection as shown in J but repressed by felodipine, fasudil, imatinib, and caspofungin are shown in a Venn diagram. L KEGG analysis for those 706 genes that were upregulated by SARS-CoV-2 infection but commonly suppressed by felodipine, fasudil, imatinib, and caspofungin as described in K is shown

Article Snippet: For treatment, fasudil (HY-10341, MCE), caspofungin (HY-17006, MCE) or imatinib (HY50948, MCE) was dissolved in PBS, and felodipine predissolved in DMSO was then diluted with sesame oil (#S25527, Shanghai Yuanye).

Techniques: Infection, Staining, Expressing, Enzyme-linked Immunosorbent Assay, RNA Sequencing, Gene Expression

Journal: Cellular & molecular immunology

Article Title: A SARS-CoV-2-specific CAR-T-cell model identifies felodipine, fasudil, imatinib, and caspofungin as potential treatments for lethal COVID-19.

doi: 10.1038/s41423-023-00985-3

Figure Lengend Snippet: Fig. 6 A SARS-CoV-2-specific CAR-T-cell model identifies felodipine, fasudil, imatinib, and caspofungin as effective treatments for SARS-CoV-2- infected Syrian hamsters. We demonstrated the feasibility of producing SARS-CoV-2-S CAR-T cells that have immunological profiles similar to those of T cells from COVID-19 patients in the presence of the S protein. This CAR-T-cell model can be used to screen drugs to suppress SARS- CoV-2-induced cytokine release. Among the drugs screened, felodipine, fasudil, imatinib, and caspofungin are effective in protecting against SARS-CoV-2-induced lethal inflammation in Syrian hamsters

Article Snippet: For treatment, fasudil (HY-10341, MCE), caspofungin (HY-17006, MCE) or imatinib (HY50948, MCE) was dissolved in PBS, and felodipine predissolved in DMSO was then diluted with sesame oil (#S25527, Shanghai Yuanye).

Techniques: Infection

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Angiotensin II Type 2 Receptor Modulates Synovial Macrophage Polarization by Inhibiting GRK2 Membrane Translocation in a Rat Model of Collagen-Induced Arthritis.

doi: 10.4049/jimmunol.2000561

Figure Lengend Snippet: FIGURE 2. The expression of AT1R/AT2R and the number of M1/M2 SMs are upregulated in RA patients’ synovial tissues. (A and B) IF staining is shown for CD68 (green); AT1R, AT2R, TGM2, and iNOS (red) and DAPI (blue). The single colors were merged to a combined picture. Original mag- nification 3400. (C) The AT1R and AT2R expression in PBMCs of RA patients was evaluated by ELISA after cell lysis. (D) Correlation analyses of AT1R and AT2R expression on PBMCs with inflammatory index were demonstrated by Pearson correlation test. Data are presented as mean 6 SEM, with 15 control and 32 RA patients. *p , 0.05, **p , 0.01 versus control group.

Article Snippet: AT2R agonist CGP42112 (catalog no. HY-12405), AT2R antagonists PD123319 (catalog no. HY-10259) and etanercept (HY-108847) were obtained from MedChemExpress.

Techniques: Expressing, Staining, Enzyme-linked Immunosorbent Assay, Lysis, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Angiotensin II Type 2 Receptor Modulates Synovial Macrophage Polarization by Inhibiting GRK2 Membrane Translocation in a Rat Model of Collagen-Induced Arthritis.

doi: 10.4049/jimmunol.2000561

Figure Lengend Snippet: FIGURE 3. AT2R activation attenuates the clinical symptoms of CIA rats. Sprague Dawley rats were immunized with CCII to establish the CIA model. The onset of clinical symptoms of arthritis was ∼day 14 after the first injection. CIA rats were randomly divided into different groups as follows: untreated CIA rats receiving an intra-articular injection of same volume of normal saline twice a week; CGP42112 group receiving treatment with intra-articular injection of CGP42112 (5, 10, and 20 mg/kg) twice a week; etanercept group receiving 3 mg/kg twice a week by i.p. injection was used as a positive control treatment. (A) The photographs of representative secondary paws from each group are presented at day 34. (B) Body weight was measured every 4 d. (C) Secondary paw swelling was checked measuring paw volume every 4 d. Data are shown as mean 6 SEM (n = 6). *p , 0.05, **p , 0.01 versus control group; #p , 0.05, ##p , 0.01 versus CIA group.

Article Snippet: AT2R agonist CGP42112 (catalog no. HY-12405), AT2R antagonists PD123319 (catalog no. HY-10259) and etanercept (HY-108847) were obtained from MedChemExpress.

Techniques: Activation Assay, Injection, Saline, Positive Control, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Angiotensin II Type 2 Receptor Modulates Synovial Macrophage Polarization by Inhibiting GRK2 Membrane Translocation in a Rat Model of Collagen-Induced Arthritis.

doi: 10.4049/jimmunol.2000561

Figure Lengend Snippet: FIGURE 4. AT2R activation improves arthritic joints histopathology and modulates spleen T cells subset and serum cytokines level of CIA rats. Un- treated CIA rats received an intra-articular injection of same volume of normal saline twice a week; CGP42112 group received treatment with intra-articular injection of CGP42112 (5, 10, and 20 mg/kg) twice a week. Etanercept, 3 mg/kg twice a week, was used as a positive control treatment. By doing this, we tried to choose an optimal concentration of CGP42112 for our further investigation. (A) Representative micrographs of H&E-stained histological sections of the joints are shown. The histological sections show the synoviocytes (S), the pannus (P), the inflammatory cells (I), the bone (B), the cartilage (C), and articular cavity (A). Original magnification 3200. (B) Representative x-ray images are shown. Arrow a represents soft tissue swelling, Arrow b represents monosaccate or multisaccate destruction of bone, Arrow c shows narrowing of the joint space. (C) Representative tables of the cytokines level in serum. (D) The level of production of the T cell cytokines IL-4, IL-17, and IFN-g. Data are mean 6 SEM (n = 5 or 6). **p , 0.01 versus control group; #p , 0.05, ##p , 0.01 versus CIA group.

Article Snippet: AT2R agonist CGP42112 (catalog no. HY-12405), AT2R antagonists PD123319 (catalog no. HY-10259) and etanercept (HY-108847) were obtained from MedChemExpress.

Techniques: Activation Assay, Histopathology, Injection, Saline, Positive Control, Concentration Assay, Staining, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Angiotensin II Type 2 Receptor Modulates Synovial Macrophage Polarization by Inhibiting GRK2 Membrane Translocation in a Rat Model of Collagen-Induced Arthritis.

doi: 10.4049/jimmunol.2000561

Figure Lengend Snippet: FIGURE 5. AT2R activation regulates AT1R expression and SM polarization. CIA rats were treated with intra-articular injection of CGP42112 (20 mg/kg) twice a week and etanercept (3 mg/kg) twice a week by i.p. injection. Untreated CIA rats receiving an intra-articular injection of same volume of normal saline twice a week were used as control. (A) The number of SM infiltration. (B and C) The population of CD11b+AT1R+SM and CD11b+AT2R+SM. (D and E) The population of CD11b+CD86+ M1-type SM and CD11b+CD163+ M2-type SM were directly determined by flow cytometry in freshly digested synovial tissue single-cell suspension. Data are mean 6 SEM (n = 6). **p , 0.01 versus control group; #p , 0.05, ##p , 0.01 versus CIA group.

Article Snippet: AT2R agonist CGP42112 (catalog no. HY-12405), AT2R antagonists PD123319 (catalog no. HY-10259) and etanercept (HY-108847) were obtained from MedChemExpress.

Techniques: Activation Assay, Expressing, Injection, Saline, Control, Cytometry, Suspension

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Angiotensin II Type 2 Receptor Modulates Synovial Macrophage Polarization by Inhibiting GRK2 Membrane Translocation in a Rat Model of Collagen-Induced Arthritis.

doi: 10.4049/jimmunol.2000561

Figure Lengend Snippet: FIGURE 6. AT2R activation inhibits NO expression. The NO expression was used to evaluate the optimal dose concentration of AT2R agonist CGP42112 and AT2R antagonist PD123319 for further experiments. (A) BMDMs were pretreated with different concentrations of AT2R agonist CGP42112 for 2 h before incubation with LPS (1 mg/ml, 24 h). Data are mean 6 SEM (n = 6). **p , 0.01 versus control group. #p , 0.05, ##p , 0.01 versus LPS (1 mg/ml) group. (B) Half an hour before AT2R agonist CGP42112 treatment, we give different concentration of AT2R antagonist PD123319 to block the effect of CGP42112. IL-4 (10 ng/ml, 24 h) was used as positive control. Data are mean 6 SEM (n = 6). **p , 0.01 versus control group; ##p , 0.01 versus CGP42112 (1026 M) group; $$p , 0.01 versus PD123319 (1028 M) group.

Article Snippet: AT2R agonist CGP42112 (catalog no. HY-12405), AT2R antagonists PD123319 (catalog no. HY-10259) and etanercept (HY-108847) were obtained from MedChemExpress.

Techniques: Activation Assay, Expressing, Concentration Assay, Incubation, Control, Blocking Assay, Positive Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Angiotensin II Type 2 Receptor Modulates Synovial Macrophage Polarization by Inhibiting GRK2 Membrane Translocation in a Rat Model of Collagen-Induced Arthritis.

doi: 10.4049/jimmunol.2000561

Figure Lengend Snippet: FIGURE 7. AT2R activation regulates AT1R expression and BMDM polarization. (A) BMDMs were pretreated with CGP42112 or PD123319 and then incubated with LPS (1 mg/ml, 4 h), and Western blot was used to detect the AT1R/AT2R expression after AT2R activation. (B) BMDMs were pretreated with CGP42112 or PD123319 and then incubated with LPS (1 mg/ml, 24 h), and BMDMs polarization marker expression were detected by Western blot after AT2R activation. Data are shown as mean 6 SEM (n = 4). *p , 0.05, **p , 0.01 versus control group; ##p , 0.01 versus LPS group; $p , 0.05, $$p , 0.01 versus LPS plus CGP group; $p , 0.05, $$p , 0.01 versus LPS plus CGP group.

Article Snippet: AT2R agonist CGP42112 (catalog no. HY-12405), AT2R antagonists PD123319 (catalog no. HY-10259) and etanercept (HY-108847) were obtained from MedChemExpress.

Techniques: Activation Assay, Expressing, Incubation, Western Blot, Marker, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Angiotensin II Type 2 Receptor Modulates Synovial Macrophage Polarization by Inhibiting GRK2 Membrane Translocation in a Rat Model of Collagen-Induced Arthritis.

doi: 10.4049/jimmunol.2000561

Figure Lengend Snippet: FIGURE 8. AT2R activation suppresses the phosphorylation of ERK1/2 and inhibits NF-kB signaling. BMDMs were pretreated with CGP42112 or PD123319 and then incubated with LPS (1 mg/ml, 4 h). (A) Western blot analysis depicting the phosphorylated and total protein forms of p38 MAPK, ERK1/2, and JNK in MAPK signaling. (B) The phosphorylation of IkB and NF-kB p50/p65 subunit nuclear expression was also detected by Western blot. Data are presented as mean 6 SEM (n = 4). *p , 0.05, **p , 0.01 versus control group; #p , 0.05, ##p , 0.01 versus LPS group; $$p , 0.01 versus LPS plus CGP group.

Article Snippet: AT2R agonist CGP42112 (catalog no. HY-12405), AT2R antagonists PD123319 (catalog no. HY-10259) and etanercept (HY-108847) were obtained from MedChemExpress.

Techniques: Activation Assay, Phospho-proteomics, Incubation, Western Blot, Expressing, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Angiotensin II Type 2 Receptor Modulates Synovial Macrophage Polarization by Inhibiting GRK2 Membrane Translocation in a Rat Model of Collagen-Induced Arthritis.

doi: 10.4049/jimmunol.2000561

Figure Lengend Snippet: FIGURE 9. AT2R activation inhibits GRK2 membrane translocation and promotes the coexpression with p-ERK1/2. BMDMs were pretreated with CGP42112 or PD123319 before the addition of LPS (1 mg/ml, 4 h). (A) The membrane protein levels of GRK2 were determined by flow cytometry (n = 5). (B) The total, membrane, and cytoplasm protein levels of GRK2 were determined by Western blot analyses (n = 4). (C) Confocal imaging of GRK2 translocation in BMDMs, Original magnification 3400. (D) LPS-stimulated BMDMs with CGP42112 or PD123319 were immunoprecipitated using an anti-GRK2 Ab and subjected to Western blotting to visualize ERK1/2 to evaluate its association with the GRK2 in cytoplasm. Coimmunoprecipitation (IP): anti-GRK2; immunoblot (IB): p-ERK1/2 and GRK2. (n = 3). **p , 0.01 versus control group; #p , 0.05, ##p , 0.01 versus LPS group; $p , 0.05, $$p , 0.01 versus LPS plus CGP group.

Article Snippet: AT2R agonist CGP42112 (catalog no. HY-12405), AT2R antagonists PD123319 (catalog no. HY-10259) and etanercept (HY-108847) were obtained from MedChemExpress.

Techniques: Activation Assay, Membrane, Translocation Assay, Cytometry, Western Blot, Imaging, Immunoprecipitation, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Angiotensin II Type 2 Receptor Modulates Synovial Macrophage Polarization by Inhibiting GRK2 Membrane Translocation in a Rat Model of Collagen-Induced Arthritis.

doi: 10.4049/jimmunol.2000561

Figure Lengend Snippet: FIGURE 10. The possible underlying mechanism of AT2R anti- inflammatory role on SM. The coexpression between p-ERK1/2 and GRK2 in cytoplasm is increased after AT2R activation, and the de- creased dissocation of p-ERK1/2 fails to promote IkB phosphorylation. Thus, the activation of NF-kB is downregulated because of low phosphorylation of IkB, reduced nuclear translocation, and a low level of inflammatory cytokines.

Article Snippet: AT2R agonist CGP42112 (catalog no. HY-12405), AT2R antagonists PD123319 (catalog no. HY-10259) and etanercept (HY-108847) were obtained from MedChemExpress.

Techniques: Activation Assay, Phospho-proteomics, Translocation Assay