Research Paper Volume 1, Issue 4 pp 402—411

Figure 1. miR146a/b expression increases in senescent HCA2 fibroblasts. (A) Northern blot analysis of total RNA prepared from proliferating (P), quiescent (Q), damage (bleomycin)-induced senescent (DS) and replicatively senescent (RS) HCA2 cells. We analyzed 10 μg of RNA from P, Q and DS cells, but 5 μg of RNA from RS cells. After separation and transfer to membranes, the blots were probed for miR-146a. Equal RNA loading was confirmed by probing for the small RNA species U6. Values for the percentage of cells incorporating bromodeoxyuridine (% BrdU) or expressing the sensecence-associated beta-galactosidase (% SA-β-gal) are indicated below each lane. (B) Northern blot analysis of RNA from DS cells. Cells were harvested for RNA at the days indicated after cells were induced to senesce by bleomycin. The blot was initially probed for miR-146a, then stripped and reprobed for miR-146b. The proliferation levels (% BrdU) and % cells that express the SA-β-gal are indicated. (C) Northern blot analysis of replicatively senescencing cells. Cells were harvested at the PD (population doubling level) indicated below the figure. The proliferation levels (% BrdU) and % cells that express the SA-β-gal are indicated. (D) Northern blot analysis of cells treated with H₂O₂ (0.1 mM for 2 h) or infected with the lentivirus expressing oncogenic RASV12. Cells were harvested for RNA at the indicated days after treatment. The proliferation levels (% BrdU) and % cells that express the SA-β-gal are indicated.