Research Paper Volume 12, Issue 14 pp 14791—14807

Figure 4. MiR-185 overexpression induces cellular senescence in primary human fibroblast cells. (A) Immunofluorescence and fluorescence in situ hybridization (IF-FISH) were performed in early passage HFF cells stably infected with viruses encoding empty vector (miR-Neg), shPOT1 or miR-185. (B) Quantification of the percentage of cells in (A) with more than 3 TIFs. (C) CDKN1A mRNA levels were detected by qRT-PCR. (D) CDKN2A mRNA level were detected by qRT-PCR. (E) HFF cells were stably infected with viruses encoding empty vector (miR-Neg), shPOT1, miR-185, miR-185 plus GFP, or mIR-185 plus POT1 respectively, miR-185 levels were detected by qRT-PCR. Relative levels of miR-185 were normalized using the U6 RNA. (F) POT1 mRNA levels were detected by qRT-PCR. Relative expression of the POT1 gene was normalized using GAPDH level. (G) HFF cells stably infected with viruses encoding empty vector (miR-Neg), shPOT1, miR-185, miR-185 plus GFP, or mIR-185 plus POT1 respectively, were stained for β-galactosidase activity (SA-β-gal). (H) Quantification of the percentage of cells in (G) positive for SA-β-gal staining. Error bars indicate standard deviations (n=3). P values were determined by Student’s t-test. ***P<0.001.