Decreased Jumonji Domain-Containing 3 at the Promoter Downregulates Hematopoietic Progenitor Kinase 1 Expression and Cytoactivity of T Follicular Helper Cells from Systemic Lupus Erythematosus Patients

Figure 3

Effects of JMJD3 downregulation on Tfh cells from 3 normal controls. (a, b) Relative JMJD3 and HPK1 protein levels in normal Tfh cells transfected with control-siRNA or JMJD3-siRNA were measured by western blot analysis after stimulation. β-Actin was used as endogenous control. (c–f) Relative JMJD3 (c), H3K27me3 (d), H3K4me3 (e), MLL1, MLL2, MLL3, and MLL4 (f) enrichments at the HPK1 promoter in normal Tfh cells transfected with control-siRNA or JMJD3-siRNA were quantified by combining ChIP and qPCR assays after stimulation. Results were normalized to input DNA (total chromatin). (g) The proliferation activities of normal Tfh cells transfected with control-siRNA or JMJD3-siRNA were assessed by MTT assays after stimulation. (h) The relative concentrations of IL-21, BAFF, IFNγ, and IL-17A in the supernatants of normal Tfh cells transfected with control-siRNA or JMJD3-siRNA were examined using ELISA after stimulation. (i) After stimulation, some normal Tfh cells transfected with control-siRNA or JMJD3-siRNA were cocultured with autologous B cells for 8 days, whereafter the concentrations of IgM, IgG1, IgG2, and IgG3 in the supernatants were detected by ELISA. The data from every control-siRNA group member were set as 1, and the multiple of every JMJD3-siRNA group member relative to its homologous control-siRNA group member was calculated.

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