Fig. 3: Mutation of SUMOylation sites abrogates G9a-mediated proliferation.
From: SUMOylation of G9a regulates its function as an activator of myoblast proliferation
a, b Cellular proliferation was measured by bromodeoxyuridine (BrdU) uptake in vector control, pBABE-G9a, and pBABE-G9a4KR cells. The percentage of BrdU+ cells is shown. c Lysates from proliferating pBABE, pBABE-G9a, and pBABE- G9a4KR cells were analyzed by western blotting using Cyclin D1, Cyclin A, and β-actin antibodies. The band intensities for Cyclin A and Cyclin D1 relative to β-actin were quantified in each cell line. The value in pBABE cells was given an arbitrary value of 1. d Flow cytometric analysis of pBABE-, pBABE-G9a-, and pBABE-G9a4KR-expressing cells synchronized at the G1/S boundary using hydroxyurea (HU). Cells were released into the growth media for 2 and 4 h before cell cycle analysis. Percentage of cells in G0/G1, S, and G2/M phases are indicated. e Lysates from proliferating pBABE-G9a and pBABE-G9a4KR cells transfected with pCS2 or SENP1 were analyzed by western blotting using Cyclin D1, Cyclin A, and β-actin antibodies. The expression of Cyclin A and Cyclin D1 was quantified relative to β-actin and is shown in the bar graph below. Control cells were given an arbitrary value of 1. f, g BrdU staining and percentage of BrdU+ cells was analyzed in proliferating pBABE, pBABE-G9a, and pBABE-G9a4KR cells transfected with either empty vector pCS2 or SENP1. Error bars indicate mean ± S.D. Scale bar = 100 μm. Significance was determined using Student’s t-test (*p < 0.05, **p< 0.01, ***p < 0.001).
