Figure 1

BRAT1 expression is required for optimal proliferation and viability. (A) NC (nonspecific shRNA) and Sh (selected BRAT1 knockdown cells) were selected and cloned from U2OS and HeLa parental cells after transfection with 4 different shRNA against BRAT1 mRNA. The expression of BRAT1 was confirmed by immunoblot (inserts). Actin protein was used as internal control. The number of live cells (trypan blue negative) was directly counted at indicated days. (B) 4 different BRAT1 knockdown HeLa cells (sh3, sh8, sh15, and sh17) were cultured for 3 days (upper panel) and indicated days (bottom panel), then cell proliferation was measured using the MTT assay. (C) Both control and knockdown U2OS cells were treated with NCS (1 μg/ml) or hydroxyurea (HU, 5 μM), then cultured for 24 h. Cells were fixed and stained with propidium iodide (PI). DNA profile was analyzed by a flow cytometry. (D) Both control and BRAT1 knockdown cells were cultured for indicated times without changing media, and then subjected to apoptosis analysis using AnnexinV/PI double stain. Apoptosis and necrosis were expressed by percentage from total cells in dot plot graphs. Data are mean of three independent experiments. **Student’s t-test: p < 0.01.