Fig 2: DYRK1A regulated NFATC1 to facilitate glioma cell migration. A, T98G cells were transfected with DYRK1A and NFATC1 expression vectors alone or simultaneously. Transwell assay showed that co-expression of DYRK1A and NFATC1 greatly increased T98G cell migration. B, T98G cells were transfected with vectors knocking down DYRK1A and/or NFATC1. Simultaneous knockdown of DYRK1A and NFATC1 decreased T98G cell migration. C, T98G cells were transfected with DYRK1A or NFATC1 mutant expression vector alone or simultaneously. Transwell assay showed that co-expression of DYRK1A and NFATC1 mutants marginally increased T98G cell migration. D, MTT assay showed that T98G cell viability was not affected by expression levels of DYRK1A and/or NFATC1. E, MTT assay showed that T98G cell viability was not affected by knockdown of DYRK1A and/or NFATC1. F, MTT assay showed that T98G cell viability was not affected by expression of DYRK1A and/or NFATC1 mutants. Values represent means ± SD, n = 3

Fig 3: Direct NR2F6 binding to the minimal Il17a promoter is dependent on its DNA- and ligand-binding domains. (A) GST-NR2F6 binds to min Il17a (NFAT o3) of the Il17a promoter region in electromobility shift assays. (B) Strep-ChIP assay in Jurkat cells transfected with NR2F6 or NFATc1 Strep-tagged proteins and the Il17a promoter region (1.3 kb). Jurkat cells were either rested (medium) or stimulated overnight (ON) with PMA/Iono, and the cell lysates were immunoprecipitated with an anti-Strep antibody and amplified with a primer pair for the minimal Il17a promoter. The data shown are derived from two independent experiments demonstrating consistent results. (C) ChIP analysis of naive Nr2f6+/+ or Nr2f6-/- CD4+ T cells either left unstimulated or stimulated under Th0 or Th17 conditions for 3 days, then stimulated with aCD3 ON, crosslinked with formaldehyde and immunoprecipitated with anti-NR2F6. The bound DNA was amplified by qRT-PCR using a primer pair from the minimal Il17a promoter locus. (D) The schematic depicts a series of mutant NR2F6 proteins with either a defective DNA binding domain (C112S; S89E) or ligand-binding domain (LL/AA; E383Stop). (E) In vitro-derived NR2F6 (from Origen) and NR2F6-GST but not the DNA (NR2F6-C112S) and ligand-binding domain mutants (NR2F6 E383Stop; LL/AA) bound to the min Il17a (NFAT o3) in EMSAs (F) a COUP-TF oligonucleotide was used as the positive control. The binding of NR2F6 is dependent on the presence of a supershift NR2F6 antibody. One of three independent experiments is shown. (G) Sequence of the mouse Il17a promoter region containing the min Il17a (NFAT o3) (underlined) (NFAT core sequence is highlighted in green), as well as the two HREs (confirmed for ROR?t [46];) and the core motif of the second half site of the NR2F6 binding sites (highlighted in red), are shown. min Il17a (NFAT o3): 5'-CCTC ATTTGAGGAT GGAATCTTTA CTCAAA -3'; min Il17a (NFAT mo3): 5'-CCTC ATTAAGGGAT GGAATCTTTA CTCAAA -3'. (H) EMSA analysis revealed that binding of NR2F6 was only disturbed when using min. Il17a (NFAT mo3), suggesting that this TGA represents a NR2F6 in vivo binding half site. NR2F6 can also bind to the RORE sites [46] within the min. Il17a promoter. One of two independent experiments is shown.

Fig 4: Predicted protein–protein interaction network of fibronectin–integrin signaling molecules and osteoclast differentiation markers. Genes encoding fibronectin–integrin signaling molecules include Itgb3, Itba5, Fn1, Pxn, Ptk2, Prkca, and RhoA, as verified by this study. Osteoclast differentiation markers include the downregulated profile (Nfatc1, Ctsk, DC-stamp, Rela, Camk2a, Traf6, Mmp9, and Acp5) verified in Figure 3. The connection edges shown are of high confidence (0.700), with the thicker connection line indicating the highest confidence (0.900). The network proteins were clustered into two groups using the k-means method

Fig 5: MicroRNA (MiR)–223 is required for calcineurin-NFAT (nuclear factor of activated T cells)/ETS2 (a member of the E26 transformation-specific sequence [ETS] domain family) pathway. A, MiR-223 levels in the hearts of Cre, F/F, and knockout (KO) mice subjected to sham or severe transverse aortic constriction (sTAC) for 3 weeks (n=6). B, MiR-223 levels in the hearts of ETS2F/F and KO mice crossed with calcineurin transgenic mice (n=8). C, MiR-223 levels in neonatal rat ventricular myocytes (NRVMs) transfected with small interfering RNA (siRNA) control (siNC) or siRNA targeting ETS2 (siETS2) and then treated with phenylephrine (PE) for 24 hours (n=6). D, Chromatin immunoprecipitation (ChIP) analysis using an ETS2-specific antibody to detect ETS2 binding to miR-223 promoter in control NRVMs or NRVMs treated with PE for 6 hours (n=4). E, MiR-223 levels in NRVMs transfected with siNC or siRNA targeting NFATc1 (siNFATc1; n=6). F, Luciferase activity in HEK-293 cells transfected with a luciferase reporter plasmid harboring a full-length miR-223 promoter, along with expression plasmids of ETS2, ETS2 lacking DNA binding domain (ETS-?DBD), and NFATc1 (n=6). G, ChIP analysis using an ETS2-specific antibody or a GFP (green fluorescent protein) antibody to detect ETS2 or NFATc1 binding to the miR-223 promoter. HEK-293 cells were transfected with a miR-223 luciferase reporter plasmid, along with expression plasmids of ETS2 and NFATc1-GFP. A total of 48 hours after transfection, cells were collected for ChIP analysis (n=4). H, MiR-223 levels in indicated NRVMs. NRVMs were infected with an adenovirus expressing constitutively activated calcineurin (AdCnA) or a control adenovirus (AdLacZ). Before harvest, calcineurin-overexpressing NRVMs were treated with cyclosporin A (CsA) or U0126 for 6 hours (n=6). I, Quantification of the relative cardiomyocyte cross-sectional area. NRVMs were transfected with antagomir control (antagomir-NC) or miR-223 antagomir and then infected with AdCnA for 48 hours (n=50). J, Protein synthesis in NRVMs treated as in (I; n=6). *P<0.05; **P<0.01; ***P<0.001. Ctrl indicates control; IgG, immunoglobulin G; and ns, not significant.