C/EBP-b Regulates Cell Death Figure 4. ER stress triggers caspase3–independent cell death, which is attenuated by LAP. (A) F10.9-3 cells (26106) in 9 cm plates were challenged either with tunicamycin for 24 & 48 h or with murine IFN-c and TNF-a (1000 IU/ml and 100 ng/ml, respectively, 48 h; used as a positive control for caspase 3-mediated apoptosis). Cell extracts were analyzed by immunoblotting using antibodies to cleaved caspase 3. (B) F10.9-3 cells in 9 cm plates were cultured for 24 h with or without doxycycline (Doxy). The cells were then treated with diluent (Ctrl) or tunicamycin (Tm, 0.25 mg/ml, 24 h). Clarified culture supernatants of the cell cultures (50 ml) were subjected to immunoblotting with antibodies to HMGB1. (C) Total cellular proteins of the cells from (B) were isolated at 24 h and subjected to immunoblot analysis with the indicated antibodies. Each of the immuno- blot shown is a representative of three replicate experiments. doi:10.1371/journal.pone.0009516.g004 in a second group of mice. The inhibition of endogenous LAP by over-expression of LIP led to a significant attenuation of tumor growth as determined at day 13 (P,0.005, N = 9). In contrast, Figure 5. LAP increases B16 melanoma tumor mass. (A) C57/BL6 mice were inoculated with F10.9-4 cells. Dominant negative LIP was induced by doxycycline in the drinking water. Mice were euthanized at day 8 or 13, and the tumors were removed and weighed. Data are presented as the average6SD of 9 tumors. Inset: Photograph of representative tumors. (B) C57/BL6 mice were inoculated with F10.9-3 cells. LAP was induced by doxycycline in the drinking water. Mice were euthanized at day 8 or 13, and the tumors were removed and weighed. Data are presented as the average6SD of 10 tumors. Inset: Photograph of representative tumors. doi:10.1371/journal.pone.0009516.g005 over-expression of LAP significantly increased the tumor mass by fourfold over a 5 day period (P,0.001, N = 10; Figure 5). To distinguish between the possible effects of LAP on tumor cell proliferation and cell death we stained tumor sections with the proliferation marker Ki-67 [37]. The percent of Ki67 (dividing) cells in tumors isolated at day 8 was not significantly affected by over- expression of LIP (P = 0.570, N = 4; Figure 6A, top panels). In accordance with our results in vitro (Figure 3), over-expression of LAP in the tumor resulted in a statistically significant reduction of Ki67-positive cells (P,0.03, N = 4; Figure 6A, bottom panels). Hence, the increase in the tumor mass upon induction of LAP must have been due to reduction in cell death, which apparently PLoS ONE | www.plosone.org 6 March 2010 | Volume 5 | Issue 3 | e9516