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. 2022 Sep 18;26(20):5135–5149. doi: 10.1111/jcmm.17539

FIGURE 1.

FIGURE 1

miR‐486 promotes cellular senescence and apoptosis and inhibits proliferation and the cell cycle at the S and G2/M phases in CMFs in vitro. (A) Immunofluorescence images of isolated CFs and TGF‐β‐induced transdifferentiation CMFs (Vimentin‐EGFP, α‐SMA‐Cy3). (B) The expression of α‐SMA and Col1a1 was significantly increased in CMFs, which confirmed successful transdifferentiation to CMFs. (C) miR‐486 is expressed in both CFs and CMFs. (C1) and (C2) The expression level of miR‐486 in CFs and CMFs is shown as a ratio to the U6 and Ct values. (D) β‐gal histological staining showed that the number of β‐gal‐positive CMFs was increased significantly in the miR‐486‐treated group. (D1‐4) Representative images of the mimic NC‐, miR‐486‐, inhibitor‐ and inhibitor NC‐treated groups. (D5) Quantitative analysis of (D1‐4). (E) The flow cytometry quantitative assay double confirmed that the number of β‐gal‐positive CMFs was increased significantly in the miR‐486‐treated group. (E1) Representative pattern of flow cytometry analysis. (E2) Quantitative analysis of (E1). (F) The CCK‐8 assay showed that the proliferation of CMFs in the miR‐486‐treated group was significantly lower than that in the mimic NC and control groups. (G) Flow cytometry analysis of apoptosis showed that the number of apoptotic cells in the miR‐486‐treated CMFs was significantly higher than that in the mimic NC‐treated CMFs. (G1‐4) Representative patterns of the mimic NC‐, miR‐486‐, inhibitor‐ and inhibitor NC‐treated groups. (G5) Quantitative analysis of (G1‐4). (H) Cell cycle analysis with flow cytometry revealed that miR‐486 overexpression in CMFs was able to promote cell cycle inhibition at the S and G2/M phases. (H1‐4) Representative patterns of the mimic NC‐, miR‐486‐, inhibitor‐ and inhibitor NC‐treated groups. (H5) Quantitative analysis of (H1‐4). (I) The double staining analysis of cellular senescence and apoptosis in miR‐486‐treated CMFs suggests that in vitro, miR‐486 overexpression is able to promote cellular senescence and apoptosis of CMFs and induce more senescent CMFs than apoptotic CMFs. Importantly, most miR‐486‐treated senescent CMFs do not undergo apoptosis simultaneously. n = 3.