Journal: Cellular & molecular immunology
Article Title: A novel cytokine consisting of the p40 and EBI3 subunits suppresses experimental autoimmune arthritis via reciprocal regulation of Th17 and Treg cells.
doi: 10.1038/s41423-021-00798-2
Figure Lengend Snippet: Fig. 5 The p40-EBI3-Fc protein suppressed Th17 cells via regulation of IL-12Rb1 and gp130 receptor signaling. A Splenocytes from C57BL/6 mice were cultured with 10 ng/mL IL-6 in the presence or absence of the p40-EBI3-Fc protein (1 or 10 μg/mL). After 1 h, protein was isolated from the cultured cells. p-STAT3 Tyr705, STAT3, p-STAT5, STAT5, and GAPDH were analyzed by western blotting. Representative western blots from one of three independent experiments are shown (bottom panel); the results are depicted as the mean ± SD of three independent experiments per group (right). B Splenocytes isolated from C57BL/6 mice were stimulated with IL-6 in the presence or absence of the p40-EBI3-Fc protein (1 or 10 μg/mL) for 15 min. CD4+ T cells expressing pSTAT3 Tyr705, pSTAT3 Ser727, or pSTAT5 were then analyzed by flow cytometry. C A IL-12Rβ1 and gp130 overexpression vector was transfected into the Ba/F3 cell line using a P4 Primary cell 4D-nucleofectorTM X kit. The proliferation of Ba/F3 cells was determined with a [3H] thymidine incorporation assay under p40-EBI3-Fc (0.01–1 μg/mL) treatment. D IL-12Rβ1- and gp130-expressing Ba/F3 cells were stimulated with p40-EBI3-Fc for 1 h. p-STAT5, STAT5, and GAPDH in the cell lysates were analyzed using western blotting. Representative western blots from one of three independent experiments are shown; the results represent the mean ± SD of three independent experiments per group (right). E, F Alterations in IL-17 production in anti-CD3-stimulated murine splenic CD4+ T cells induced by p40-EBI3-Fc treatment and transfection with IL-12Rβ1-, gp130-, or WSX1-siRNA. Th17 cell expression was determined by FACS, and IL-17 expression was evaluated by ELISA. Bars show the mean ± SD of three independent experiments per group (*p < 0.05, **p < 0.01, ***p < 0.001).
Article Snippet: Mouse splenocytes were stimulated with 0.5 μg/mL anti-CD3 for 12 h. The cells were then harvested and cotransfected with IL-12 receptor-specific (IL-12Rβ1 and WSX1 or IL-12Rβ1 and gp130; 100 nM) siRNAs (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in accordance with the manufacturer’s protocol.
Techniques: Cell Culture, Isolation, Western Blot, Expressing, Cytometry, Over Expression, Plasmid Preparation, Transfection, Thymidine Incorporation Assay, Enzyme-linked Immunosorbent Assay
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![Fig. 5 The p40-EBI3-Fc protein suppressed Th17 cells via regulation of IL-12Rb1 and <t>gp130</t> receptor signaling. A Splenocytes from C57BL/6 mice were cultured with 10 ng/mL IL-6 in the presence or absence of the p40-EBI3-Fc protein (1 or 10 μg/mL). After 1 h, protein was isolated from the cultured cells. p-STAT3 Tyr705, STAT3, p-STAT5, STAT5, and GAPDH were analyzed by western blotting. Representative western blots from one of three independent experiments are shown (bottom panel); the results are depicted as the mean ± SD of three independent experiments per group (right). B Splenocytes isolated from C57BL/6 mice were stimulated with IL-6 in the presence or absence of the p40-EBI3-Fc protein (1 or 10 μg/mL) for 15 min. CD4+ T cells expressing pSTAT3 Tyr705, pSTAT3 Ser727, or pSTAT5 were then analyzed by flow cytometry. C A <t>IL-12Rβ1</t> and gp130 overexpression vector was transfected into the Ba/F3 cell line using a P4 Primary cell 4D-nucleofectorTM X kit. The proliferation of Ba/F3 cells was determined with a [3H] thymidine incorporation assay under p40-EBI3-Fc (0.01–1 μg/mL) treatment. D IL-12Rβ1- and gp130-expressing Ba/F3 cells were stimulated with p40-EBI3-Fc for 1 h. p-STAT5, STAT5, and GAPDH in the cell lysates were analyzed using western blotting. Representative western blots from one of three independent experiments are shown; the results represent the mean ± SD of three independent experiments per group (right). E, F Alterations in IL-17 production in anti-CD3-stimulated murine splenic CD4+ T cells induced by p40-EBI3-Fc treatment and transfection with IL-12Rβ1-, gp130-, or <t>WSX1-siRNA.</t> Th17 cell expression was determined by FACS, and IL-17 expression was evaluated by ELISA. Bars show the mean ± SD of three independent experiments per group (*p < 0.05, **p < 0.01, ***p < 0.001).](https://pub-med-unpaywalled-images-cdn.bioz.com/pub_med_ids_ending_with_2759/pm34782759/pm34782759__page9_image1.jpg)