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Fig. 5 | BMC Molecular and Cell Biology

Fig. 5

From: Dlx5 and Dlx6 can antagonize cell division at the G1/S checkpoint

Fig. 5

Dlx proteins limit entry into S-phase. a, b Proliferating C2C12 cells were transfected and seeded onto poly-D-Lysine-coated coverslips and DNA synthesis was detected 24-h post transfection by incorporation of EdU for 4 h. a Representative pictures taken with a 40x objective. Yellow arrows point to Dlx-positive cells that have incorporated EdU during the incubation time. b Quantitation of the labeling index (LI), defined as the ratio of EdU-positive nuclei to total cells. Bars represent the average LI, +/− SEM from 3 experiments, with at least 200 Dlx-positive cells counted per experiment. **p < 0.01 (unpaired t-test). c, d C2C12 cells, treated as above, were labeled with EdU for various times to measure the growth fraction (GF) and cell cycle length. Each time point represents a minimum of three experiments. LI is plotted over time for (c) Dlx5- and (d) Dlx6-expressing cells. Slopes of the initial linear increases, as determined by linear regression analysis, were compared in Prism using the “Comparison of Regression Lines” function. Neither Dlx5 nor Dlx6 significantly altered the rate of EdU incorporation, compared to the surrounding Dlx-negative cells (Dlx5 p = 0.07; Dlx6 p = 0.14). GF was set as the mean of all points following the initial linear increase. The elevations of the GF reached by Dlx-positive and Dlx-negative cells were compared using the “Comparison of Regression Lines” function in Prism, and both proteins were found to significantly alter the GF, compared to the surrounding Dlx-negative cells (Dlx5, Dlx6 p < 0.0001). e, f Transfected C2C12 cells were selected over a period of 4 days then 8 × 105 cells were seeded into 6 cm plates. Cells were collected for flow cytometric analysis and extra cells were re-plated at the original density every 24 h. e Representative flow cytometry histogram at 48 h post selection. Scaling on both axes is linear. f Quantitation of flow cytometry data. Bars represent the mean of all experiments (n = 4) +/− SEM; G1/G0 (red), S (orange), G2/M (green). Both Dlx5- and Dlx6-expressing populations contained a reduced proportion of cells in S-phase, with a compensatory increase in G1. **p < 0.0001, unpaired t-test

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