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. 2012 Jan 5;7(1):e29907. doi: 10.1371/journal.pone.0029907

Figure 3. Downregulation of Nkx2-1 affects expression of proliferation-related genes in mouse lung epithelial cells.

Figure 3

(A) Western blot analysis of Nkx2-1 downregulation by shRNA in MLE15 cells using the Nkx2-1 antibody 07-601 (Millipore-Upstate). Two bands between 40–50 kD are detected in mouse MLE15 cells and E17.5 lung. Densitometry analyses show a significant downregulation of Nkx2-1 major bands in shRNA transduced cells. Nkx2-1 downregulation by shRNA also reduces the level of minor bands of higher molecular weight (*, asterisks) (B) Representative flow cytometry analyses of DNA content and cell cycle progression in the same cells; n = 3. (C) Relative number of cells in each stage of the cell cycle. (D) Cell count analysis of MLE15 cells transduced with empty vector or Nkx2-1shRNA. 5000 cells were plated and grown in standard conditions for up to 4 days. Attached cells were trypzinized and counted; n = 6 (E) Real time RT-PCR analyses of Nkx2-1 mRNA expression levels in Nkx2-1 shRNA or empty vector transduced cells; n = 3; similar analyses were performed for selected targets; n = 6. Error bars represent s.e.m.; (*) p≤0.05.