inducibly over-express the dominant-negative LIP, thereby inhibiting endogenous LAP activity, as well as other clones that inducibly over-express LAP. Our findings suggest that C/EBP-b has a key role in regulating the transition from protective to death promoting UPR; LAP attenuates and LIP augments cell death, and LAP contributes to tumor progression by attenuating ER stress and subsequent cell death. Results LAP Attenuates and LIP Augments ER Stress-Triggered Cell Death To study the role of LAP to LIP ratio in ER stress-triggered cell death, we used the specific inducers of ER stress tunicamycin (Tm) and thapsigargin (Tg) and murine B16 melanoma clones that inducibly express either LAP or LIP [34]. Clone F10.9-3 constitu- tively expresses the Tet repressor and carries a vector with a bidirectional inducible promoter encoding wt LAP and GFP. Immunoblotting with an antibody directed against the common C- terminus of the various C/EBP b isoforms revealed that doxycycline induced only LAP in Clone F10.9-3 (Figure S1). Similarly, Clone F10.9-4 expressed only LIP, which is the dominant-negative form of LAP (Figure S1). Initially, we triggered ER stress in un-induced cells and compared the progression of cell death and the expression of endogenous LAP and LIP. Cell detachment and death started 4–6 h after addition of tunicamycin, as determined quantitatively by staining of the remaining attached cells with Crystal Violet (Figures 1A & B, solid lines). Basal LAP and LIP were undetectable by immunoblotting. As previously reported [18], LAP expression was apparent already at 3 h, whereas LIP appeared at 6 h (Figure 1C). Thus, initiation and further progression of cell death was temporally associated with endogenous LIP levels. To further evaluate the role of LAP and LIP in determining the cell fate under ER stress, we studied the impact of their over- expression on the progression of cell death, as determined by Neutral Red staining. Over-expression of LIP in the F10.9-4 cells prior to addition of tunicamycin abolished the 4 h ‘‘grace period’’, leading to a statistically significant increase of cell death by 4 h (P,0.009, N = 4), as well as at all later time points (P,0.01, N = 4; Figure 1A, dashed line). No cell death was seen upon LIP expression in the absence of ER stress (data not shown). Over- expression of LAP had no significant effect in the first 4 h, but significantly attenuated cell death at all other time points (P,0.01– 0.03; N = 4; Figure 1B, dashed line). These observations demonstrated that LAP attenuates and LIP augments ER stress- triggered cell death. Since both LAP and LIP were induced past the first 5 h, we evaluated their combined effect on cell viability by siRNA knockdown. Silencing of both LAP and LIP expression significantly augmented the tunicamycin-triggered death of F10.9- 3 cells as determined by viability staining with Neutral Red (P,0.0001; N = 9; Figure 1D). Because LIP lacks a trans- activation domain, it probably augmented cell death by inhibiting ER stress-induced LAP. Microscopic observation of Crystal Violet–stained cultures further demonstrated that LIP over-expression augmented thapsi- gargin or tunicamycin–triggered cell death at 24 h (Figure 2A). In contrast, over-expression of LAP attenuated thapsigargin or tunicamycin–triggered cell death, as determined at 24 and 48 h, respectively (Figure 2B). The impact of C/EBP-b on tunicamy- cin–induced cell death was further determined quantitatively by viability staining with Neutral Red. Over-expression of LIP significantly augmented tunicamycin–induced cell death (P,0.0001, N = 9), whereas over-expression of LAP significantly attenuated cell death (P,0.0001, N = 9; Figure 2C, D, C/EBP-b Regulates Cell Death respectively). A similar increase in ER stress-triggered cell death was obtained in three other independent F10.9 LIP-expressing sub-clones following induction with doxycycline. Similarly, ER stress–triggered cell death was attenuated in four other indepen- dent F10.9 LAP-expressing sub-clones following doxycycline (data not shown). The enhancement of cell death by LIP was dose– dependent and apparent upon induction with $100 ng/ml doxycycline (Figure S2A). Similarly, the attenuation of cell death was dose–dependent and apparent upon induction of LAP with $100 ng/ml doxycycline (Figure S2B). Microscopic evaluation of Annexin V staining of tunicamycin- challenged cultures revealed lack of apoptotic cells within 24 h of treatment (Figure S3A). Rather, tunicamycin triggered cell rounding and detachment from the plate. The detached cells were dead or dying, as determined by propidium iodide staining (Figure S3B). Some of the tunicamycin-treated cells remained attached to the plate and although they were propidium iodide negative, they appeared to be dead, as they were condensed and rounded (Figure S3C, left panels). Unlike control cells, the LAP– over expressing F10.9-3 cells survived the tunicamycin treatment, they remained attached to the plate and appeared viable (Figure S3C, right panels). Furthermore, they propagated normally after removal of the ER stress triggers (data not shown). Based upon these observations we concluded that the ER stress-induced LAP attenuated cell death, whereas LIP augmented cell death, probably by inhibiting or re-directing the pro-survival activity of LAP. To determine how general is the role of C/EBP-b in ER stress, we studied the impact of C/EBP-b over-expression and knockout in additional cell types. Over-expression of LAP in human 293T cells by transfection with an expression vector designed to encode LAP only (pcLAP, Figure S4A) rendered the cells significantly more resistant to tunycamycin-triggered cell death (P,0.0007, N = 4, Figure S4B). In contrast, over-expression of LIP in human 293T cells by transfection with an expression vector encoding only the LIP open reading frame (pcLIP, Figure S4A) rendered the cells significantly more sensitive to tunycamycin-triggered cell death (P,0.003, N = 4, Figure S4B). Knockdown of C/EBP-b in human HeLa cells by siRNAs specific to C/EBP-b led to efficient silencing of its expression at 48 h, as determined by RT-PCR and immunoblotting with an antibody directed against a common C-terminal peptide of C/ EBP-b (Figure S4C; left and right panels, respectively). The silencing of C/EBP-b increased the sensitivity of HeLa cells to ER stress–triggered cell death (Figure S4D), leading to a statistically significant decrease in the cell counts (P,0.0003, N = 4, Figure S4E). Over-expression of LAP in human HeLa cells by transfection with pcLAP rendered the cells significantly more resistant to tunicamycin-triggered cell death (P,0.001, N = 6, Figure S4F). In contrast with all other experiments, over- expression of LIP in HeLa cells by pcLIP also rendered the cells more resistant by 13% to tunicamycin-triggered cell death (P,0.002, N = 5). Yet, all other results suggest a general role for C/EBP-b in modulating ER stress–triggered cell death. Attenuation of Cell Death by LAP Prevails Over Its Inhibitory Effect on the Cell Cycle To study the mechanism by which LAP and LIP regulate the cell fate upon ER stress, we first determined the impact of LAP over-expression on the cell cycle. As previously reported [35], treatment of the B16 melanoma clone F10.9-3 with tunicamycin abolished DNA synthesis, leading to cell cycle arrest, as determined by flow cytometry of propidium iodide–stained cells (Figure 3 compare the two left panels). Over-expression of LAP in F10.9-3 cells for 24 h prior to treatment with tunicamycin or PLoS ONE | www.plosone.org 2 March 2010 | Volume 5 | Issue 3 | e9516