Figure 6.

Proviral Integration site for Moloney murine leukemia virus 1 (Pim-1) promotes lung fibroblast premature senescence. A: non-idiopathic pulmonary fibrosis (IPF) adult lung fibroblasts were infected with control lentiviral vector or lentiviral vector carrying the human PIM1 gene. Total protein was isolated in three independent Western blot experiments. Representative images are shown and band density was quantified (**P < 0.01 vs. the Lenti-Control group). B: Lenti-Control and Lenti-PIM1 expressing cells were treated for 3 h or 24 h with 3 µM TCS before total protein isolation and Western blot analysis. Representative images are shown and band density for three independent experiments was quantified (**P < 0.01, ***P < 0.001 vs. the indicated group). C and E: qPCR analysis from three independent experiments comparing gene expression between Lenti-Control and Lenti-PIM1 fibroblasts (*P < 0.05, **P < 0.01, ***P < 0.001 vs. the Lenti-Control group). D: Lenti-Control and Lenti-PIM1 expressing fibroblasts were cultured for 0 and 4 days and then stained for DAPI and quantified using automated imaging software. Representative images are shown for the number of DAPI objects (nuclei) on day 4. Quantification of three independent experiments plots the number of cells in each field of view, for each cell line, relative to the number of cells in each field of view immediately after the cells attached (day 0) (*P < 0.05 vs. the Lenti-Control group). The scale bar represents 200 µm. F: senescence-associated β-galactosidase (SA-β-gal) staining comparing Lenti-Control vs. Lenti-PIM1 lung fibroblasts as they progress through cell culture passages 4–8. Representative images comparing fibroblasts at passage 8. Data are automated quantification of SA-β-gal positive cells relative to the total number of cells in each field of view from three independent experiments (*P < 0.05, ****P < 0.0001 vs. the Lenti-Control group). Scale bar represents 100 µm.