Fig 2: Camk2d and Ppp3r2 are direct targets of miR-146a in vivo. (a-e) Immunohistochemical staining and quantitative analysis of the percentage of Camk2d, Ppp3r2, Nfatc1 and Nfatc2-positive chondrocytes in knee joint cartilage of 3- and 12-month-old WT or miR-146a KO mice. n=8 per group. Scale bars, 100 µm. (f-h) Immunohistochemical staining and quantitative analysis of the percentage of Camk2d and Ppp3r2-positive chondrocytes in knee joint cartilage harvested from the indicated groups. n=8–9 per group. Scale bars, 100 µm. Data are the mean±S.D. *P<0.05, **P<0.01 (Mann–Whitney test). NS, not significant between the indicated groups

Fig 3: STIM2 regulates the expression and nuclear translocation of transcription factor NFAT1. a qRT-PCR analysis of the expression of transcription factors in MDA-MB-231 cells with STIM2 (top) or STIM1 (bottom) knockdown (SH) or overexpression (OE). b qRT-PCR analysis of the expression of NFAT1 in MCF7 cells with STIM2 knockdown or overexpression. c Western blots (left) and quantification (right) of NFAT1 protein levels in MDA-MB-231 cells with STIM2 or STIM1 knockdown or overexpression. d Western blots (left) and quantification (right) of NFAT1 protein levels in MCF7 cells with STIM2 knockdown or overexpression. e Western blots (left) and quantification (right) showing NFAT1 levels in the nuclear extracts of a panel of breast cancer cell lines. f Immunofluorescent staining (× 100) of NFAT1 in MDA-MB-231 cells with STIM2 knockdown or overexpression. One-way ANOVA was used to compare independent groups. Data shown are means ± SD of at least 3 independent experiments. *P < 0.05

Fig 4: TSF alleviated NFATC2 nuclear translocation in AGEs-stimulated primary mice podocytes. (a) Representative immunofluorescence staining and quantitative analysis of NFATC2 (scale bars, 50 µm; 30 cells evaluated per experiment, n = 3 experiments). Nuclei were stained with DAPI. (b) Immunofluorescence staining for NFATC3 (scale bars, 50 µm). The data were expressed as the mean ± SEM. #P < 0.05, ##P < 0.01 vs. BSA group; *P < 0.05, **P < 0.01 vs. AGEs group; ?P < 0.05, ??P < 0.01 vs. AGEs + TSF group. Statistically analyzed via a one-way ANOVA with Dunnett's correction.

Fig 5: Effects of NFAT1 on TGF-ß1 expression and secretion. a Ingenuity Pathway Analysis summary. Using IPA, we identified downstream molecules of NFAT1, including TGF-ß1, a key regulator of EMT. b Results of ELISA showing the secretion of TGF-ß1 with knockdown (SH), overexpression (OE), or rescue of NFAT1 and STIM2. c Results of dual-reporter luciferase assay showing NFAT1-dependent activation of the TGF-ß1 promoter. Reporter gene constructs were transfected into HEK 293 T cells, and luciferase activity was measured after 48 h. d Chromatin immunoprecipitation of NFAT1 on the TGF-ß1 promoter. After cells were lysed, proteins were cross-linked and immunoprecipitated with an anti-NFAT1 antibody. The cross-linking was reversed, and PCR was performed. One of 3 separate experiments is shown. e Signaling pathway formed by STIM2, NFAT1, and TGF-ß1 was showed. T tests or one-way ANOVA were used to compare independent groups. Data shown are means ± SD from at least 3 independent experiments. NC, control. *P < 0.05