Hes-6 increases proliferation and E2F-1 transcription in T47D cells. (a) Expression of Hes-6 mRNA in T47D-control (bar 1) and T47D-Hes-6 cells (bar 2) cells (three samples/bar). (b) Proliferation assay performed with MTS kit (Promega) after 5 days' incubation ± E2 on synchronized, polyclonal T47D-control/Hes-6 cells. T47D-control cells without (bar 1) or with (bar 2) E2, T47D-Hes-6 cells without (bar 3) or with (bar 4) E2 (12 samples/bar). (c) Relative E2F-1 mRNA levels in T47D-control (bar 1) and T47D-Hes-6 (bar 2) cells (three samples/bar) in nonsynchronized cells. (d) E2F-1 (upper lane) and Hes-6 protein (lower lane) in synchronized T47D-control/Hes-6 cells, measured with Western blot. Graphs represent protein intensity of E2F-1 Western blot, analyzed with Biorad software; T47D-control (bar 1) and T47D-Hes-6 at 0 hours (bar 2) in the absence of E2, T47D-control (bar 3), and T47D-Hes-6 at 24 hours (bar 4) in the presence of E2. (e) Relative hASH-1 mRNA levels in T47D-control (bar 1) and T47D-Hes-6 (bar 2) cells (three samples/bar) in nonsynchronized cells. Graphs are shown as relative values to bars 1 (100%) of each graph. All graphs are presented as mean values ± SD. Significance is presented as *P < 0.05; **P < 0.005; and ***P < 0.001.