C/EBP-b Regulates Cell Death Figure 3. Cell cycle analysis of F10.9-3 cells. F10.9-3 cells (26106) were cultured in 9 cm plates with or without doxycycline to induce LAP. The cells were then challenged with tunicamycin, collected, permeabilized using 70% ethanol at 220uC, stained with propidium iodide and analyzed using flow cytometry. The graphs are representatives of three replicate experiments. doi:10.1371/journal.pone.0009516.g003 thapsigargin did not reverse the cell cycle arrest. In fact, LAP lowered the extent of cell proliferation in control cells, reducing the number of cells in S phase by about 25% and increasing the percentage of cells in the G0/G1 phase (Figure 3, top panels). The observed LAP-mediated inhibition of cell proliferation was consistent with a previous study on the impact of LAP (LAP-2) on cell cycle progression [26]. Hence, we concluded that the pro- survival activity of LAP must have been the outcome of its effect on cell death rather than on the cell cycle. Furthermore, the attenuation of cell death by LAP under stress prevailed over its inhibitory effect on unstressed cell proliferation. We therefore focused on the impact of LAP and LIP on cell death mechanisms. LAP Attenuated Necrotic Cell Death by Lowering CHOP Expression Although tunicamycin triggered rounding, detachment and death of the F10.9 cells, we were unable to detect either annexin V-positive cells (Figure S3A) or cleaved caspase 3 in the cell extracts (Figure 4A). This result was also in accordance with the absence of sub G0 cell population, as revealed by flow cytometry (Figure 3). Rather, we found that ER stress triggered the release of High-Mobility Group Box 1 (HMGB1) to the culture supernatant, indicating a mechanism involving necrotic cell death [36]. Furthermore, over-expression of LAP attenuated the release of HMGB1 (Figure 4B). Therefore, we concluded that LAP attenuates the ER stress-triggered necrosis of F10.9 cells. The lack of annexin V staining minimizes the possibility that necrosis was initiated by an early stage of a caspase 3-independent apoptotic mechanism. To further study the mechanism by which LAP modulates ER stress, we measured the effect of its over-expression on the early and late ER stress markers Binding Protein (BiP; GRP78) and CHOP in tunicamycin-treated F10.9-3 cells. Immunoblot analysis revealed that over-expression of LAP did not lower the ER stress–induced chaperone BiP. In contrast, LAP reduced the level of the death executor CHOP (Figure 4C). These results indicated that LAP does not affect the early protective phase of the UPR. Rather, LAP attenuates the late, death-promoting phase of the UPR. LAP Promotes Tumor Progression by Attenuating Cell Death To study the role of LAP in tumor progression, we inoculated mice subcutaneously either with F10.9-4 cells or with F10.9-3 cells and induced LIP or LAP expression, respectively, by including doxycycline (Doxy; 1 mg/ml) in the drinking water. Palpable tumors were detected in the mice after 8 days and differences in tumor mass were apparent by day 13. Therefore tumors in one group of mice were isolated and examined at day 8 and at day 13 PLoS ONE | www.plosone.org 5 March 2010 | Volume 5 | Issue 3 | e9516