Figure 4
SAHA inhibits TGF-β1-induced cell proliferation. (a) Phase-contrast photographs of MRC5 treated with TGF-β1 and/or SAHA for 24 h (10x magnification). The experiment was performed three times (in triplicate per condition). (b) Cell proliferation of MRC5 was evaluated by cell countingassay. Results are presented as the mean number ± SD of MRC5 at 24 hours of incubation for each condition. The same amount of cells was used to start each incubation time, control and treated cells. Three independent experiments were carried out with three replicatesfor each experimental condition. One star (*) indicates a p ≤ 0.05, while two stars (**) indicate a p ≤ 0.001, compared to control group; and ## indicates a p ≤ 0.01, compared to TGF-β1-treated group, two-tailed Student’s test. (c) Effect of TGF-β1 and/or SAHA on the viability of MRC5 was measured based on cell index by the xCELLigence system (Roche). Cell Index (CI) was recorded every 30 minutes. Each trace at each concentration was an average of 3 replicates. Data are normalized to the time of treatments addition to cell cultures. One star (*) indicates a p ≤ 0.05, while two stars (**) indicate a p ≤ 0.001, compared to control group; and ## indicates a p ≤ 0.01, compared to TGF-β1-treated group, two-tailed Student’s test. (d) Apoptotic/necroticeffects of the different treatment on MRC5 were evaluated by Annexin V/PI staining. Graphs showing the percentage of viable, apoptotic and necrotic cells treated SAHA and/or TGF-β1 for 24 h. The experiment was performed three times. (e) Effect of TGF-β1 and/or SAHA on the cell cycle of MRC5 cells. After 24 h of treatment, cells were stained with propidium iodide to determine the DNA content. The percentages of the cells in each phase of the cell cycle were determined by flow cytometric analysis. The experiment was performed three times.
