C/EBP-b Regulates Cell Death Figure 8. Vascularization of control and LIP–or LAP–expressing tumors. Sections of control and LIP–expressing tumors of similar sizes, isolated at day 8, (top panels) were stained with CD31 antibodies, and nuclei were counter-stained with Hoechst 33258. Sections of control and LAP– expressing tumors of similar sizes, isolated at day 8 (bottom panels), were stained with CD31 antibodies. The photographs are representatives of sections obtained from 3 tumors from each one of the four treatment groups. doi:10.1371/journal.pone.0009516.g008 stress-triggered cell death in vitro. Since the death executor TRIB3 is induced by CHOP, our finding that LAP attenuated and LIP augmented the expression of TRIB3 in the tumor (Figure 10A) suggests that LAP promotes tumor progression by attenuating CHOP-mediated cell death. By performing knockdown and over- expression studies in two other human cell types (Figure S4) we demonstrated the general role of LAP and LIP in modulating the resistance to ER stress-triggered cell death. A previous study demonstrated that C/EBP-b mice (lacking both LAP and LIP) are completely refractory to generation of skin tumors upon exposure to a carcinogen [8]. Our findings further show that C/ EBP-b promotes the progression of an established tumor by attenuating cell death, implicating ER stress in the process. Our findings suggest that changes in the LAP-LIP ratio control the transition from the early, protective UPR to its late, death– promoting phase. Others [18] and we observed that ER stress initially triggered the expression of LAP, whereas extended ER stress led to expression of both LAP and LIP (Figure 1). C/EBP-b was found to be constitutively bound to the CHOP promoter at the common C/EBP-ATF4 site, inhibiting CHOP expression in rat cells [45]. Our findings that LAP attenuated the expression of CHOP and its downstream target TRIB3 (Figures 4C &10A) suggest that LAP rather than LIP is the constitutive transcriptional inhibitor of CHOP. Thus, LIP may relieve the transcription inhibition of LAP by forming a futile LAP-LIP heterodimer and thereby triggering cell death. Alternatively or additionally, such a LAP-LIP heterodimer may trigger cell death in a manner analogous to the proposed action of the LAP-CHOP heterodimer [5]. Regardless of the exact mechanism, this study demonstrates that LAP inhibits the death-promoting phase of the UPR whereas in most cases LIP promotes ER stress–triggered cell death. Establishing the impact of LAP and LIP on gene expression under ER stress will further clarify the mechanism by which LAP attenuates cell death. Materials and Methods Ethics Statement The Weizmann Institute Animal Care and Use Committee approved all studies in mice, according to institutional guidelines and Israeli law. Cells and Reagents The murine B16 melanoma clones, F10.9-3 and F10.9-4, were derived from the parental cell line B16-F10 (ATCC CRL-6475) and were kindly provided by M. Revel and J. Chebath (Weizmann Institute of Science) [34]. Human cervical carcinoma HeLa cells (CCL-2.1) and human embryonic kidney cells (HEK 293T) were obtained from the American Type Culture Collection (ATCC), Manassas, VA. Cell culture media were supplemented with 10% FBS and antibiotics (DMEM-10 and MEM-10) and cells were grown in humidified 8% CO2 incubators at 37uC. Expression of LAP and LIP was induced in the F10.9-3 and F10.9-4 cells, respectively by doxycycline (Doxy, 1 mg/ml, 24 h) unless otherwise stated. ER stress was elicited by treatment with tunicamycin (0.75 mg/ml, 24 h) or thapsigargin (50 nM, 48 h) unless otherwise PLoS ONE | www.plosone.org 9 March 2010 | Volume 5 | Issue 3 | e9516