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. 2017 Feb 8;7:29. doi: 10.3389/fcimb.2017.00029

Figure 3.

Figure 3

EV-D68 infection prevents cell exit from G0/G1 into S phase and promotes G2/M to G0/G1 transition. (A) RD cells were serum-starved for 24 h and then mock-infected (mock) or infected with EV-D68 Fermon strain (infected) at an MOI of 0.8. After 2 h of virus adsorption, the cells were treated with medium without FBS for 18 h, followed by medium containing 10% FBS. Top panel: The cell cycle profiles were determined by flow cytometry at 0, 18, 24, and 30 h post-infection. Bottom panel: The histograms show the percentage of cells in each phase of the cell cycle. (B) G0/G1 and S phase-related cell cycle proteins were analyzed by western blot analysis. RD cells were mock-infected (m) or infected with EV-D68 Fermon strain at an MOI of 0.8 (i) for 0, 16, 20, 24, and 28 h. Histone is shown as a loading control. Results are representative of three independent experiments. (C) At 24 h post-infection, mRNA levels of CDK2, cyclinE1, CDK4, CDK6, and cyclinD were evaluated in mock infected (mock) and EV-D68 infected (infected) cells by quantitative real-time PCR. The results are standardized to GAPDH and normalized to 1.0 in mock-infected cells. (D) RD cells were treated with 25 ng/mL nocodazole or control medium for 24 h, infected with EV-D68 Fermon strain at an MOI of 0.8 or mock for 2 h and then re-treated with 25 ng/mL nocodazole for synchronization. Top panel: Cell-cycle profiles were determined by flow cytometry. Low panel: The histograms show the percentage of cells in each phase of the cell cycle. (E) G2/M phase-related proteins were detected by western blot analysis. Results are representative of three independent experiments. (F) At 24 h post-infection, mRNA levels of CDK1 and cyclinB1 were assessed in mock-infected (mock) and EV-D68 infected (infected) cells by quantitative real-time PCR. The results are standardized to GAPDH mRNA and normalized to 1.0 in mock-infected cells. (G) At 28 h post-infection, the protein concentration of CDK4, CDK6, cyclinD1, CDK2, cyclinE1, CDK1, cyclinB1, and histone were assessed in mock-infected (mock) and EV-D68 infected (infected) cells by Elisa kit. The results are standardized to Histone (his). The results indicate the mean ± S.D of three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001.