C/EBP-b Regulates Cell Death Figure 6. Ki-67 and H&E staining of tumor sections. (A) Sections of control F10.9-4 tumors and dominant negative C/EBP-b LIP–expressing F10.9-4 tumors of similar sizes, isolated at day 8, were immunostained with an antibody against Ki-67, and nuclei were counter-stained with Hoechst 33258 (top left and right panels, respectively). Percent Ki67-positive nuclei were determined by counting sections from 4 control and 4 LIP-expressing tumors (71–94 nuclei/field, middle top panel). Sections of control F10.9-3 tumors and LAP-expressing F10.9-3 tumors of similar sizes, isolated at day 8 (bottom left and right panels, respectively), were prepared and stained as above. Percent Ki67-positive nuclei were determined by counting sections from 4 control and 4 LAP-expressing tumors (60–107 nuclei/field, middle bottom panel). Representative images of sections from the four tumors are shown. Bar = 0.1 mm. (B) Sections of control F10.9-3 tumors and LAP-expressing F10.9-3 tumors of a similar size, isolated at day 8, were stained with hematoxylin and eosin. The block arrows indicate necrotic areas. Bar = 0.1 mm. The photograph is a representative of sections from 5 tumors. doi:10.1371/journal.pone.0009516.g006 overcame the anti-proliferative effect of LAP. Indeed, necrotic areas were abundant in the tumor sections (Figure 6B, block arrows). Therefore, we focused our study on the impact of LAP and LIP on the extent of tumor cell death. Immunostaining of tumor sections isolated at day 8 revealed a rather low frequency (#3%) of cleaved caspase 3-positive cells in control tumors and in tumors expressing either LAP or LIP. Furthermore, expression of either LIP or LAP had no statistically significant effect on the fraction of the cleaved caspase 3-positive cells (P = 0.64, N = 3 and P = 0.52, N = 3 for LIP and LAP, respectively; Figure 7). Hence, similarly to the in vitro study (Figure 4), spontaneous B16 melanoma cell death in vivo was probably mediated by a caspase-3 independent mechanism. LAP Attenuates Hypoxia–and Nutrient Deprivation–Triggered Cell Death LAP could attenuate tumor cell death indirectly by inducing angiogenic factors or by modulating intrinsic tumor cell functions. Staining of frozen tumor sections with antibodies to the endothelial cell marker CD31 did not reveal a significant effect of LAP on the extent of tumor vascularization (Figure 8). Hence, we concluded that LAP attenuated mechanisms that trigger tumor cell death or rendered the cells more resistant to such mechanisms. We therefore studied the impact of LAP on B16 cell survival upon triggering ER stress by the tumor-related stressors hypoxia and nutrient deprivation. To this end, we employed extreme hypoxic conditions (1% oxygen) or media completely lacking nutrients (serum-free Earle’s Balanced Salt Solution (EBSS)) in a series of in vitro studies. As expected, hypoxia or nutrient deprivation led to massive ER stress-triggered cell death (Figure 9). Over-expression of LAP significantly attenuated the extent of cell death (P,0.0001, N = 4 for hypoxia; P,0.0002, N = 4 for starvation; determined by measuring the relative Crystal Violet staining intensity; Figure 9). These results were in accordance with the impact of LAP on the specific inducers of ER stress tunicamycin and thapsigargin. PLoS ONE | www.plosone.org 7 March 2010 | Volume 5 | Issue 3 | e9516