Fig. 2. The Effect of TGFβ on DNA synthesis in HEPM cells.

(A) To investigate the effect of TGFβ on DNA synthesis in HEPM cells, serum starved synchronized HEPM cells were treated with 10ng/ml of TGFβ1, 2 and 3 as well as transiently transfected with a full length human c-Myc cDNA, (pcDNA3.3 c-Myc) and and small hairpin RNA (shRNA) c-Myc, pRetrosuper-sh-cMyc (Addgene, MA) for 24 h followed by addition of 30μM 5-ethynyl-2-deoxyuridine (EdU). TGFβ1, 2, 3 and c-Myc treatment stimulated cell cycle progression, measured by EdU incorporation as DNA synthesis progresses, compared to negative control (0.5% FBS). 10% FBS was used as a positive control to show stimulation of DNA synthesis. TGFβ3 had the greatest effect on EdU incorporation compared to TGFβ1 and 2. (B) The percentage of cells incorporated with EdU was measured and values were presented as means ±SD from at least three measurements. Using BD Biosciences FACSArray, a significant proportion of cells were found to occupy distinct cell cycle phases including G0/G1, S, and G2/M within the selected boxed region. Larger boxed region identifies all proliferating cells as determined by their levels of DNA synthesis as assessed by PI within the G0/G, S and G2/M phases of the cycle. The smaller inset box includes the cells that are incorporated with EdU or S phase cells. Here, we only show the cells that are EdU positive cells in the S phase. A p-value of ≤ 0.05 was considered significant. The one-way ANOVA indicated that the values differ significantly across the treatment groups. All three TGFβ treatment groups and c-Myc also differed significantly from the positive (10% FBS) and negative (0.5% FBS) control groups, (p ≤ 001, as indicated by **, while between the controls, the different was also significant p≤0001, as indicated by *).