Fig 2: Further influence of RCAN1.4 silencing in HK-2 cells and NRK-49F cells.A Representative blots of RCAN1.4, COL1a1, a-SMA, and GAPDH; B COL1a1/GAPDH ratio; C a-SMA/GAPDH ratio; D RCAN1.4/GAPDH ratio. E Representative blots of Bax, Bcl-2, cleaved-caspase3 and GAPDH; F Bax/Bcl-2 ratio; G cleaved-caspase3/GAPDH; *p < 0.05, #p < 0.05, +p < 0.01, &p < 0.01, as indicated. The results are expressed as the mean ± standard error of the mean (S.E.M.). for 3–4 independent experiments.

Fig 3: RCAN1.4 expression was down-regulated in vivo and vitro.Western blotting analysis was performed after collecting kidney lysates. A, F Representative blots of RCAN1.4, COL1a1, a-SMA, and ß-actin; B, H a-SMA/ß-actin ratio; C, G COL1a1/ß-actin ratio; D, I RCAN1.4/ß-actin ratio; E Immunohistochemistry signals of RCAN1(Scale bars, 20 µm and 50 µm). The results are expressed as the mean ± standard error of the mean (S.E.M.) for 3–4 independent experiments. *p < 0.05, as indicated.

Fig 4: sEV-miR-324 derived from osteoclasts can be delivered into MSCs and induce osteogenic differentiation(A) The expression profile of miRNAs in sEVs secreted during osteoclast differentiation. sEVs from BMMs were used as a normalization control. Red color represents higher expression, and blue color represents lower expression relative to the control. (B) ALP activity assay shows that overexpression of miR-324 promoted osteogenic differentiation; n = 3. (C and D) Relative expression levels of (C) miR-324 and (D) pri-miR-324 in MSCs cultured with sEVs from miR-324 overexpression (miR-324-sEVs) or knockdown (anti-miR-324-sEVs) osteoclasts; n = 3. (E) Cell viability evaluation of MSCs cultured with miR-324-sEVs or anti-miR-324-sEVs using a CCK-8 test at 1, 3, 5, and 7 days; n = 5. (F) Relative mRNA expression levels of Col1a1, Alpl, Sp7, and Runx2 in MSCs cultured with miR-324-sEVs or anti-miR-324-sEVs; n = 3. (G) Western blot analysis of COL1A1, RUNX2, ALP, and ß-actin in MSCs cultured with miR-324-sEVs or anti-miR-324-sEVs. (H) Representative images of alizarin red staining of MSCs cultured with miR-324-sEVs or anti-miR-324-sEVs. Scale bar represents 100 µm. (I) Quantification analysis of calcium deposit of MSCs in indicated groups; n = 5. Data in the figures represent the averages ± SD. *p < 0.05, **p < 0.01, for differences between the treatment and control groups.

Fig 5: RCAN1.4 negatively regulated the nuclear translocation of NFAT2 in renal fibrosis.A, B The protein levels of Calcineurin, NFAT2 and GAPDH; C–F The protein levels of NFAT2 and Histone H1; G The protein levels of COL1a1/GAPDH ratio; a-SMA/GAPDH ratio; RCAN1.4/GAPDH ratio; *p < 0.05, **p < 0.05, ***p < 0.05, #p < 0.05, ##p < 0.05, ###p < 0.05, $p < 0.05, &p < 0.05, as indicated. The results are expressed as the mean ± standard error of the mean (S.E.M.) for 3–4 independent experiments.