Journal: Virology

Article Title: SARS coronavirus 7a protein blocks cell cycle progression at G0/G1 phase via the cyclin D3/pRb pathway

doi: 10.1016/j.virol.2005.10.015

Figure Lengend Snippet: 7a expression blocked cell cycle progression at G0/G1 phase. (A) Expression of 7a fused with different tags induced G0/G1 cell cycle arrest. pCMV-myc, 7a/pCMV-myc, pEGFP-N1, 7a/pEGFP-N1 and 7a/pEGFP-C1 plasmids were transfected into HEK 293 cells separately. At 24h post-transfection, samples were collected and stained with propidium iodide (PI). The DNA contents of cells were measured by flow cytometry. Myc-7a (+) viewed with anti-myc antibody and EGFP (+) represented the positive cells (myc-7a or EGFP fusion protein expressed cells) in the middle column, and myc-7a (−) and EGFP (−) represented negative cells in the right column in the transfected cells. The experiments were independently repeated three times. (B) Cell cycle arrest induced by 7a expression in COS-7 and Vero cells. 7a/pCMV-myc plasmid was transfected into COS-7 and Vero cells. At 24 h after transfection, the DNA contents of cells were analyzed by flow cytometry as before. Three independent sets of experiments were repeated. (C) Cell cycle arrest induced by 7a expression at different times post-transfection. HEK 293 cells were transfected with 7a/pCMV-myc and pCMV-myc. At 24, 36, 48 and 60 h after transfection, samples were collected and analyzed by flow cytometry as before. Myc-7a positive and negative cells were showed with gray and white bars. Histogram was shown the percentages of cells at various phase of cell cycle with means ± SE for three independent sets of experiments. (D) Profile of Annexin V staining in 7a/pCMV-myc and pCMV-myc transfected cells. HEK 293 cells were transfected with 7a/pCMV-myc and pCMV-myc. At 24, 36 and 48 h post-transfection, samples were collected and analyzed by flow cytometry for the ability to bind Annexin V according to the manufacture's guidelines (Clontech).

Article Snippet: Cells were then permeabilized in 0.1% Triton X-100/PBS, incubated with anti-myc antibody (1:100) and FITC-conjugated mouse anti-IgG (1:100) (Santa Cruz), then resuspended in propidium iodide (PI, 50 μg/ml) staining solution (containing DNase-free RNase A 20 μg/ml) for 30 min in the dark.

Techniques: Expressing, Transfection, Staining, Flow Cytometry, Plasmid Preparation