C/EBP-b Regulates Cell Death Figure 9. LAP attenuates hypoxia–and nutrient deprivation–triggered cell death. (A) F10.9-3 cells (46105/well) were cultured in 6-well plates in media without (Ctrl) or with (LAP) doxycycline. The plates were then cultured for 53 h, either under normoxia (8% CO2 in air) or under hypoxia (1% O2 and 8% CO2 in nitrogen). The plates were then stained with crystal violet and photographed (left two panels, 1x; right two panels, light microscopy. The photographs are representatives of 4 cultures. Bar = 0.5 mm. Relative cell staining intensity is shown in the middle panel. The data are average6SD of measuring four fields. (B) F10.9-3 cells (36105/well) were cultured in 6-well plates in media without (Ctrl) or with (LAP) doxycycline. The plates were then cultured either in DMEM or in serum-free EBSS for 100 h and then stained and photographed as in (A). The photographs are representatives of 4 independent cultures. Bar = 0.5 mm. Relative cell staining intensity is shown in the middle panel. The data are average6SD of measuring four fields. doi:10.1371/journal.pone.0009516.g009 stated. Antibodies to C/EBP-b (C-19) and CHOP (B-3) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); the TRIB3 antibody (ST1032) was from Calbiochem (San Diego, CA); the Ki-67 antibody (clone TEC-3) was from DakoCytomation (Copenhagen, Denmark); the cleaved caspase 3 antibody (Asp 175) was from Cell Signaling (Danvers, MA); the HERPUD antibody was from Protein Tech Group (Chicago, IL), the rat anti-mouse CD31 (PECAM-1) and BiP/GRP78 antibodies were from BD Pharmingen, (San Diego, CA), the HMGB1 antibody (H9539) was from Sigma-Aldrich (Rehovot, Israel) and the Cy3-conjugated streptavidin, biotin-SP–conjugated AffiniPure donkey anti-rabbit IgG and biotin-SP–conjugated goat anti-mouse IgG antibodies were from Jackson ImmunoResearch (Baltimore, PA). All other reagents were purchased from Sigma-Aldrich (Rehovot, Israel). Tumor Generation and Immunohistochemistry C57/BL6 mice were inoculated subcutaneously with F10.9-3 or F10.9-4 cells (100 ml medium, containing 56105 cells mixed with 100 ml Matrigel). Doxycycline (1 mg/ml) was added to the drinking water for the induction of LAP and LIP. Mice (10 per group) were euthanized at day 8 or 13, and tumors were resected, weighed, photographed and either fixed in 10% formaldehyde or frozen before sections were prepared. The significance of differences in the tumor mass was determined by Student’s t-test of independent samples. The paraffin sections were immunostained with antibodies to Ki-67, cleaved caspase 3, TRIB3 and HERPUD, followed by incubation with biotin-conjugated antibodies and Cy3-conjugated streptavidin. Cell nuclei in tumor slices and in cultures were stained with Hoechst 33258 (5 mg/ml; excitation at 365 nm and emission at 470 nm). For CD31 immunostaining, tumor tissues were embedded in OCT and were frozen at 280uC for cryosectioning. After fixation in 100% acetone (220uC, 20 min), slides were immunostained as described above. Quantitative Measurment of the Immunostains The percentage of proliferating cells was determined by counting ki67-positive nuclei and dividing by the total number of (Hoechst 33258-stained) nuclei. The fraction of cleaved caspase 3 cells was similarly determined. The staining intensity of TRIB3 and HERPUD was determined by the histogram function of The Photoshop program (red channel) and normalized to the staining intensity of the nuclei (blue channel) in the same field. The PLoS ONE | www.plosone.org 10 March 2010 | Volume 5 | Issue 3 | e9516