Figure 1.

MiR-141 inhibits cell proliferation and induces senescence in HDFs. (A) Relative expression of microRNA in control MRC5 and MRC5 fibroblasts stably overexpressing miR-141 was determined by qRT-PCR as described in the “Materials and Methods”. (B) MiR-141 overexpression inhibits cellular proliferation. Equal number of cells were plated and harvested as indicated. Each experiment was done in triplicates, and proliferation curves were generated by plotting the number of cells against the number of days. Error bars represent ± SD *, P < 0.05. (C) Induction of cellular senescence by miR-141 overexpression was determined by SA-β-gal staining and EdU co-staining as described in the “Materials and Methods”. The number of senescent (SA-β-Gal positive), proliferating (EdU positive), and total number of cells in each culture were counted in multiple fields to determine the percentage of senescent and proliferating cells and was plotted as shown. Each experiment was done in triplicates. Error bars represent ± SD *, P < 0.05 (D) MiR-141 overexpression induces marker of DNA damage as seen by the ϒH2AX foci formation. The numbers of ϒH2AX foci/cell were quantified from multiple cells/field, and plotted as shown in the graph. Experiment was done in triplicates. Error bars represent ± SD *, P < 0.05 (E) MiR-141 overexpression inhibits proliferation and promotes senescence as determined by the western blot analysis for expression of pRb, p53, p21, and p16 in indicated set of cells. β-actin was used as the loading control. The signal for each protein was quantified using ImageJ software (NIH) and normalized to β-actin. The normalized value (relative expression) is indicated below each blot. *The relative expression values of phospho- pRb (p-pRb) and pRb are indicated as x/x.