Journal: Advanced Science

Article Title: Decreased Expression of IL‐35 and Its Receptor Contributes to Impaired Megakaryopoiesis in the Pathogenesis of Immune Thrombocytopenia

doi: 10.1002/advs.202305798

Figure Lengend Snippet: Reduced IL‐35 in patients with ITP. A) The level of plasma IL‐35 by ELISA (n = 12 in the control group, n = 26 in the ITP group). B) The concentration of IL‐35 in the bone marrow of ITP patients and healthy controls by ELISA (n = 22 in the control group, n = 10 in the ITP group). C) Expression of IL‐35 receptor subunit IL‐12Rβ2 in CD41/CD61+ cells by flow cytometry (n = 10). D) The mean fluorescence intensity of IL‐12Rβ2 on day 10 by flow cytometry (n = 10). E) Expression of IL‐35 receptor subunit gp130 in CD41/CD61+ cells by flow cytometry (n = 10). F) The mean fluorescence intensity of gp130 on day 10 by flow cytometry (n = 10). G) The protein levels of IL‐35 receptor subunits IL‐12Rβ2 and gp130 in CD41/CD61+ cells (n = 6). H) CD41 (green), IL‐12Rβ2/ gp130 (red), and DAPI (blue) staining of CD41/CD61+ cells (600×; bar: 20 µm). Data information: Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001; ns, no significant difference, using Student‘s t‐test (A, B, D, F) or ANOVA (C, E).

Article Snippet: Megakaryocytes were washed once with PBS and fixed in 4% paraformaldehyde for 15 min and then blocked with 5% bovine serum albumin at room temperature for 2 h. The cells were incubated with primary antibodies against IL‐12Rβ2 (MAB19591‐SP, R&D Systems, MN, USA), gp130 (MA5‐38467, Thermo, MA, USA), β‐tubulin (ab6046, abcam), CD41 (ab134131, abcam) overnight at 4 °C, followed by fluorescein isothiocyanate‐conjugated goat anti‐rabbit IgG antibody (ab6717, abcam) and DAPI for nuclear counterstaining (Vectashield) at room temperature for 1 h. Samples were mounted in FluorSave Reagent (Calbiochem, San Diego, CA, USA).

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Control, Concentration Assay, Expressing, Flow Cytometry, Fluorescence, Staining

Journal: Advanced Science

Article Title: Decreased Expression of IL‐35 and Its Receptor Contributes to Impaired Megakaryopoiesis in the Pathogenesis of Immune Thrombocytopenia

doi: 10.1002/advs.202305798

Figure Lengend Snippet: IL‐35 stimulates megakaryopoiesis in vitro. A) Expression of IL‐35 receptor subunit IL‐12Rβ2 in CD41/CD61+ cells from ITP patients by flow cytometry (n = 10). B) The mean fluorescence intensity of IL‐12Rβ2 on day 10 by flow cytometry (n = 10). C) Expression of IL‐35 receptor subunit gp130 in CD41/CD61+ cells from ITP patients by flow cytometry (n = 10). D) The mean fluorescence intensity of gp130 on day 10 by flow cytometry (n = 10). E) The protein levels of IL‐35 receptor subunits IL‐12Rβ2 and gp130 in CD41/CD61+ cells (n = 6). F) CD41 (green), IL‐12Rβ2/ gp130 (red), and DAPI (blue) staining of CD41/CD61+ cells (600×; bar: 20 µm). G) (Left) Representative images of megakaryocyte colonies (100×; bar: 100 µm). (Right) The number of megakaryocyte colonies (n = 3). Data information: Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001; ns, no significant difference, using ANOVA (A, C, E, F) or Student‘s t‐test (B, D).

Article Snippet: Megakaryocytes were washed once with PBS and fixed in 4% paraformaldehyde for 15 min and then blocked with 5% bovine serum albumin at room temperature for 2 h. The cells were incubated with primary antibodies against IL‐12Rβ2 (MAB19591‐SP, R&D Systems, MN, USA), gp130 (MA5‐38467, Thermo, MA, USA), β‐tubulin (ab6046, abcam), CD41 (ab134131, abcam) overnight at 4 °C, followed by fluorescein isothiocyanate‐conjugated goat anti‐rabbit IgG antibody (ab6717, abcam) and DAPI for nuclear counterstaining (Vectashield) at room temperature for 1 h. Samples were mounted in FluorSave Reagent (Calbiochem, San Diego, CA, USA).

Techniques: In Vitro, Expressing, Flow Cytometry, Fluorescence, Staining

Journal: Advanced Science

Article Title: Decreased Expression of IL‐35 and Its Receptor Contributes to Impaired Megakaryopoiesis in the Pathogenesis of Immune Thrombocytopenia

doi: 10.1002/advs.202305798

Figure Lengend Snippet: Iguratimod stimulates the expression of IL‐35 receptors on megakaryocytes. A) Expression of IL‐35 receptor subunit IL‐12Rβ2 in CD41/CD61+ cells from ITP patients by flow cytometry (n = 10). B) The mean fluorescence intensity of IL‐12Rβ2 on day 10 by flow cytometry (n = 10). C) Expression of IL‐35 receptor subunit gp130 in CD41/CD61+ cells from ITP patients by flow cytometry (n = 10). D) The mean fluorescence intensity of gp130 on day 10 by flow cytometry (n = 10). E) The protein levels of IL‐35 receptor subunits IL‐12Rβ2 and gp130 in CD41/CD61+ cells (n = 6). F) Expression of IL‐35 receptor subunit IL‐12Rβ2 in CD41/CD61+ cells from ITP mice by flow cytometry (n = 10). G) The mean fluorescence intensity of IL‐12Rβ2 in CD41/CD61+ cells from ITP mice on day 10 by flow cytometry (n = 10). H) Expression of IL‐35 receptor subunit gp130 in CD41/CD61+ cells from ITP mice by flow cytometry (n = 10). I) The mean fluorescence intensity of gp130 in CD41/CD61+ cells from ITP mice on day 10 by flow cytometry (n = 10). J) The protein levels of IL‐35 receptor subunits IL‐12Rβ2 and gp130 in CD41/CD61+ cells from ITP mice (n = 6). K) The mRNA levels of STAT genes in megakaryocytes (n = 6). L) The mRNA levels of STAT3 and STAT4 were inhibited by si‐STAT3 and si‐STAT4 in CD41/CD61+ cells (n = 6). M) The mRNA levels of IL‐12Rβ2 and gp130 in CD41/CD61+ cells (n = 6). N) The protein levels of IL‐12Rβ2 and gp130 in CD41/CD61+ cells (n = 6). (n = 6). Data information: Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, and *** p < 0.001; ns, no significant difference, using ANOVA.

Article Snippet: Megakaryocytes were washed once with PBS and fixed in 4% paraformaldehyde for 15 min and then blocked with 5% bovine serum albumin at room temperature for 2 h. The cells were incubated with primary antibodies against IL‐12Rβ2 (MAB19591‐SP, R&D Systems, MN, USA), gp130 (MA5‐38467, Thermo, MA, USA), β‐tubulin (ab6046, abcam), CD41 (ab134131, abcam) overnight at 4 °C, followed by fluorescein isothiocyanate‐conjugated goat anti‐rabbit IgG antibody (ab6717, abcam) and DAPI for nuclear counterstaining (Vectashield) at room temperature for 1 h. Samples were mounted in FluorSave Reagent (Calbiochem, San Diego, CA, USA).

Techniques: Expressing, Flow Cytometry, Fluorescence