C/EBP-b Regulates Cell Death Figure 1. Kinetics and dose-response of ER stress–induced LIP, LAP and cell death. (A) F10.9-4 cells (30,000/well) were cultured in 96-well plates with or without doxycycline (1.2 mg/ml, 24 h). Tunicamycin (0.75 mg/ml) was then added, and the cell number was determined following fixation and crystal violet staining at the indicated times. OD was converted to cell counts using a calibration curve. Data are presented as mean6SD from four replicates. (B) F10.9-3 cells (40,000/well) were cultured and treated with doxycycline and tunicamycin as described in (A). Data are presented as mean6SD from four replicates. (C) F10.9 cells (1.56106) were seeded in 9 cm plates for 24 h. Tunicamycin was then added to the cells and cell extracts were prepared at the indicated times. Extracts were resolved by SDS-PAGE and immunoblotted with an antibody directed against a common C-terminal peptide of C/EBP-b. NS: non-specific band, serving as a loading control. The blot is a representative of three replicates. (D) F10.9-3 cells were transfected with the indicated siRNA and seeded in 96 well plates (26104/well). Tunicamycin (Tm) was added after 48 h and viability was determined by Neutral Red staining after 27 h. Data are average6SD of cell counts in 9 replicates. inset: Silencing efficiency as determined by immunoblotting of C/EBP-b LAP (I) vs. actin (II) of cell extracts treated with control siRNA (1) or C/EBP-b siRNA (2). doi:10.1371/journal.pone.0009516.g001 PLoS ONE | www.plosone.org 3 March 2010 | Volume 5 | Issue 3 | e9516