Fig. 3

NFκB and NFAT1 are required to upregulate γc expression. A Expression of γc protein increases in a time-dependent manner upon PMA (12.5 ng/ml) /Iono (1 µM) stimulation. The blot is representative of four independent experiments. B Determination of γc surface expression level using FACS and (C) γc mRNA level using RT-qPCR. The relative gene expression was calculated with that of the Rpl13 housekeeping gene. D Experimental schematic of the generation of NFAT1 and NFκB KO EL4 cell lines using CRISPR/Cas9. E Generation of NFAT1 and NFκB (p65 and cRel) KO EL4 cell lines using the CRISPR/Cas9 system. WT, NFAT1 KO (#24), p65 KO, cRel KO, NFκB KO, and NFAT1/NFκB KO (#10) EL4 cells were stimulated with PMA/Iono for 16 h. NFAT1, NFκB, and γc protein levels in the indicated cell lines were analyzed using western blotting. β-actin was used as the loading control. Data are representative of four independent experiments. F Immunoblot analysis of γc expression in the presence of PMA/Iono (left); determination of membrane γc (mγc) mRNA expression using RT-qPCR (right). The relative gene expression was calculated with that of the β-actin and Rpl13 housekeeping genes. The results are the summary of four independent experiments. G γc expression in KO EL4 cell lines. KO EL4 cell lines were stimulated with PMA/Iono, and the cytokine receptor and activation marker expression was analyzed using FACS: (left) γc and CD69 expression (open histogram) assessed in activated KO EL4 cell lines and overlaid with control antibody staining (shaded histogram); (right) summary of FACS analysis for γc and CD69 expression in the presence of PMA/Iono. The results are the summary of four independent experiments. H sγc expression synergistically downregulated in NFκB KO and NFAT1/NFκB KO (#10) cells (Top). KO cells were stimulated with PMA/Iono and culture supernatants were harvested to assess soluble γc (sγc) expression. The results are the summary of four independent experiments. sγc mRNA expression was assessed using RT-qPCR (bottom). The relative gene expression was calculated with that of the Rpl13 housekeeping gene. Data are presented as the mean ± standard error of the mean of four independent experiments. * p < 0.05, ** p < 0.01, and ***p < 0.001