Fig 1: Osteoclast-specific gene expression was inhibited by RTA-408 during osteoclastogenesis. (A) Bone marrow macrophages (BMMs) were cultured to generate mature osteoclasts for 3 days with different doses of RTA-408 (0, 5, 10, 20 nM). The mRNA expression levels of calcitonin receptor (Ctr), dendritic cell-specific transmembrane protein (Dc-stamp), c-Fos, and cathepsin K (Ctsk) were measured by real-time PCR. (B) The protein expression of c-Fos and Nfatc1 was analyzed by western blotting (day 1, 3, and 5) and quantitatively analyzed (C). (D) Immunofluorescence analysis of BMMs stimulated with RANKL for 4 days to detect the expression of c-Fos and Nfatc1. (E) Quantitative analysis of the relative fluorescence signal intensity using Image J from five independent versions. The concentration of RTA-408 was 20 nM unless noted. Original scale bars: 15 μm. Data on all bar graphs are presented as the mean ± SEM(n = 3). *P < 0.05, #P < 0.005.
Fig 2: miR-22-3p mimic inhibits CD14+PBMC differentiation by targeting MAPK14. (A-C) pcDNA3.1-MAPK14 reversed miR-22-3p mimic-induced CD14+PBMCs decreased differentiation. **P<0.01 vs. pcDNA3.1 or miR-NC mimic + pcDNA3.1. &P<0.05 vs. miR-22-3p mimic + pcDNA3.1. miR, microRNA; PBMCs, peripheral blood mononuclear cells; NC, negative control; TRAP, tartrate resistant acid phosphatase; NFATC1, nuclear factor of activated T-cells; CTSK, cathepsin K.
Fig 3: 11R-VIVIT inhibits the nuclear localization and dephosphorylation of NFAT2 in RTECs in an AKI-to-CKD progression model.a Double immunofluorescence staining of NFAT2 (green) and DAPI (blue) and merged images of kidneys from the sham-operated, IRI and IRI + 11R-VIVIT treatment groups. Scale bars = 20 μm. b Quantification of the percentage of renal tubular epithelial cells with NFAT2 expression in the nucleus. *P < 0.05 vs. IRI-2d, #P < 0.05 vs. IRI-14d, &P < 0.05 vs. IRI-28d. c Protein expression of p-NFAT2 (Ser172) in the sham-operated, IRI and IRI + 11R-VIVIT treatment groups on the 2nd, 14th and 28th day. d The quantitative results of p-NFAT2 (Ser172) were normalized to GAPDH. NFAT2 nuclear factor of activated T cells 2, RTECs renal tubular epithelial cells, AKI acute kidney injury, CKD chronic kidney disease, IRI ischemia-reperfusion injury, p-NFAT2 phosphorylated nuclear factor of activated T cells 2.
Fig 4: Urolithin B inhibits the expression of osteoclast related proteins and genes. (A、F) Cell lysates were immunoblotted with antibodies against osteoclast associated proteins MMP9, CTSK, c‐fos and NFATc1. (B‐E) Quantitative analysis of concentration dependence of osteoclast associated protein. (G‐J) Quantitative analysis of time‐dependent expression of osteoclast associated protein. *p < 0.05, **p < 0.01 versus the RANKL‐induced group (without UB treatment). (K‐P) Quantitative analysis of mRNA expression of MMP9, CTSK, TRAP, ATPase, c‐fos and NFATc1. *p < 0.05, **p < 0.01 versus the RANKL‐induced group (without UB treatment)
Fig 5: Overexpression of NFATc1 partially promoted cell proliferation and EMT in miR‐338‐overexpressing NSCLC cells. A549 cells were transfected with miR‐NC or miR‐338 mimic with or without pcDNA‐NFATc1 vector. (a) The protein expression of NFATc1 was determined by western blot. (b) Cell proliferation was assessed by Brdu assay. (c) The protein expressions of CDK4, cyclin D1 and p27 were determined by western blot. (d) The expressions of E‐cadherin, Vimentin and N‐cadherin were detected by western blot assays. (e) The expressions of Snail, Slug, and ZEB1 were detected by western blot assays. All data are presented as mean ± SEM, n = 4. *p < .05, **p < .01 versus. miR‐NC; # p < .05, ## p < .01 versus. miR‐338 mimic. EMT, epithelial‐mesenchymal transition; NFATc1, nuclear factor of activated T‐cells c1; NSCLC, non‐small‐cell lung cancer
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