fluorescence microscopy. To evaluate cell morphology, cultures were fixed and stained with 5% crystal violet in 66% methanol. Total cell counts were determined by counting manually, by using the ImageJ program (NIH) or by reading the crystal violet stained cultures in 96 well plates with an ELISA reader at 570 nm. Alternatively, staining intensity was determined in photographs by the histogram function of Photoshop. For quantitative measure- ment of viable cells, cultures in 96 well plates were incubated with Neutral Red (70 mg/L in 0.1 ml DMEM-10, 37uC, 30–60 min), washed 3X with PBS, re-suspended in lysis buffer (0.1 ml) and OD was measured with an ELISA reader at 540 nm. A calibration curve was prepared by seeding known amounts of cells onto 96- well plates. After 6 h the cells were stained with Neutral Red and the OD was read as described above. For each experiment, statistical analyses were performed on the indicated number of independent experiments using Student’s t-test of the independent samples. Each independent experiment consisted of 9 replicates. Apoptosis was evaluated by staining with the rh annexin-V/FITC kit (Bender MedSystems, Vienna, Austria). Cell cycle analysis was performed by collecting the cultured cells, fixing in 70% ethanol on ice, treatment with RNase A (100 mg/ml, 30 min, 37uC), staining with propidium iodide and flow cytometry. Immunoblotting of Cellular Proteins Cells were washed three times with ice-cold phosphate buffered saline (PBS). Cell pellets were resuspended in two packed cell volume of lysis buffer (20 mM Hepes, pH 7.9, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM Phenyl Methyl Sulfonyl Flouride (PMSF), 0.5 mM dithiothreitol (DTT), 25% glycerol). The resuspended pellets were then frozen and thawed twice and kept on ice for 20 min. The lysates were centrifuged (14,000 rpm, 20 min.) and the supernatants containing the cellular proteins were collected, frozen in liquid nitrogen and stored at 280uC. Protein concentration was determined by a BCA Protein assay reagent kit (Pierce, Rockford USA) using bovine serum albumin as a standard. Protein samples were boiled in SDS-PAGE sample buffer containing 25 mM DTT, and the supernatants were resolved by gradient SDS-PAGE (7.5–15% acrylamide). Proteins were then transferred onto a nitrocellulose membrane, which was incubated with the indicated antibodies. Second antibody conjugates were visualized by the Super Signal Detection Kit (Pierce). Construction of pcLAP and pcLIP Expression Vectors To construct a mammalian expression vector that expresses only LAP (pcLAP), the complete human C/EBP-b ORF was inserted into pcDNA4 (Life Technologies, Carlsbad CA). Codon 1 (LAP*- initiating ATG) was point-mutated to CTG using Pfu Turbo DNA polymerase (Stratagene, La Jolla, CA), the forward oligo 59 TG- TGCTGGAATTCCCTGCAACGCCTGGTGG and the reverse oligo 59 ACGGGAAGCCCGCCGCCAGGCCTGCGCCGCC- GC. Codon 199 (ATG) was then point-mutated as above with the forward oligo 59 GCGGCGGCGCAGGCCTGGCGGCGGGC- TTCCCGT and the reverse oligo 59 ACGGGAAGCCCGCCGC- CAGGCCTGCGCCGCCGC to prevent expression of LIP. The LIP ORF (codons 199–346) was inserted into pcDNA4 to generate pcLIP, which expresses only the human LIP isoform of C/EBP-b. Supporting Information Figure S1 Inducible over-expression of LAP and LIP in murine B16 F10.9 clones. B16 F10.9 cells (46105/well) were cultured in 6- well plates with or without doxycycline (Doxy). (A) RT-PCR of C/ EBP-b mRNA from F10-9.3 cells. (B) GFP expression in F10 9.3 C/EBP-b Regulates Cell Death cells. Bar = 0.5 mm. (C) Dose response of LAP expression in F10 9.3 cells following 24 h treatment with increasing concentrations of doxycycline, as determined by immunoblotting. (D) Dose response of LIP expression in F10 9.4 cells, as described in (C). The gels and blots are representative results of three replicate experiments. Found at: doi:10.1371/journal.pone.0009516.s001 (1.00 MB TIF) Figure S2 LIP augments and LAP attenuates ER stress- triggered cell death in a dose-dependent manner. (A) F10.9-4 cells (26104/well) were cultured in 96-well plates with the indicated concentration of doxycycline for 24 h. Diluent (Ctrl), tunicamycin (Tm) or thapsigargin (Tg) were then added. After an additional 24 h the plates were stained with crystal violet and photographed. The photograph is representative of five replicate experiments. (B) F10.9-3 cells (1.56104/well) were cultured in 96- well plates with the indicated concentration of doxycycline for 24 h. Diluent (Ctrl), tunicamycin (Tm) or thapsigargin (Tg) was then added. After 24 h the plates were stained with crystal violet and photographed under a light microscope. Control cells were also photographed by fluorescent microscopy (top panel) to evaluate the extent of GFP and C/EBP-b induction by doxycycline. The photograph is a representative of five replicate experiments. Bar = 0.5 mm. Found at: doi:10.1371/journal.pone.0009516.s002 (9.92 MB TIF) Figure S3 Survival and death of F10.9-3 cells under ER stress. (A) F10.9-3 cells were seeded in 8 well microscopy chambers (Integrated Biodiagnostics, Munich, Germany; 40,000/well) and after 24 h challenged either with tunicamycin (0.5 mg/ml, 24 h) or with TNF-a (100 ng/ml) plus IFN-c (1000 IU/ml) for 24 h. The cells were stained for apoptosis by the rh annexin-V/FITC kit followed by Hoechst 33258. The treatment with TNF-a plus IFN- c served as a positive control for apoptosis, as determined by the annexin V/FITC staining. The photographs are representatives of four replicate studies. Bars are 0.2 mm. Insets: larger magnifica- tion to show cellular staining details. Bar = 0.05 mm. (B) F10.9-3 cells in 6-well plates were challenged with tunicamycin (1 mg/ml, 48 h). The detached cells were collected, plated and stained with propidium iodide and counter-stained with Hoechst 33258. The photograph is a representative of three replicate experiments. Bar = 0.2 mm. (C) Control (Ctrl) and doxycycline-treated F10.9-3 cells (LAP) were challenged with tunicamycin (Tm) or Thapsi- gargin (Tg) as described in Figure 2B. The cells that remained attached to the plates were stained with crystal violet and photographed. The photographs are representatives of five replicate experiments. Bar = 0.2 mm. Found at: doi:10.1371/journal.pone.0009516.s003 (4.02 MB TIF) Figure S4 Effects of LAP and LIP knockdown or over- expression on cell survival upon ER stress. (A) Human 293T cells in 6 well plates were transfected with 1 mg of control pcDNA4 (middle lane), pcLAP (left lane), or pcLIP (right lane). After 24 h cell extracts (20 mg protein) were immunoblotted with antibodies directed to the common C-terminus of C/EBP-b. N.S. is non- specific bands. The blot is a representative of three replicates. (B) Human 293T cells were transfected as in E and after 24 h were trypsin-digested and seeded in poly(D-lysine)-coated 96 well plates (20,000 cells/well). After additional 24 h the cells were either untreated (Ctrl) or challenged with tunicamycin (2 mg/ml). The plates were left for 24 h and the number of viable cells was determined by the Neutral Red assay. Percent survival represent the ratio of tunicamicyn-treated to diluent-treated cells. Data are PLoS ONE | www.plosone.org 12 March 2010 | Volume 5 | Issue 3 | e9516