C/EBP-b Regulates Cell Death Figure 10. LAP attenuates tumor ER stress. (A) Sections of control F10.9-4 tumors and dominant negative LIP-expressing F10.9-4 tumors of similar sizes, isolated at day 8 (top panels), were immunostained with antibodies against the ER stress marker TRIB3. Nuclei were counter-stained with Hoechst 33258. The average staining intensity of the red channel (immunostain) was normalized to that of the blue channel (Hoechst 33258). Data are mean6SD of sections from three tumors (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 panels), were prepared, stained and analyzed as above. Data are mean6SD of sections from 5 tumors. Representative images are shown. Bar = 0.1 mm. (B) The same experiment as in (A), except that the sections were immunostained with antibodies against the ER stress marker HERPUD. Data are mean6SD of sections from 3 and 4 tumors (middle top and bottom panels, respectively). Representative images are shown. Bar = 0.1 mm. doi:10.1371/journal.pone.0009516.g010 significance of differences in cell counts or in the staining intensity was determined by Student’s t-test on the indicated number of replicates. performed in 6-well plates with DharmaFECT 1 reagent, according to the manufacturer’s protocol. Cells were seeded in media without antibiotics for the knockdown experiments. RNAi–Mediated Knockdown siRNA pools (ON-TARGETplus), directed against murine and human C/EBP-b mRNA (Genebank accession No. NM_009883 and NM_005194, respectively) were purchased from Dharmacon RNAi Technologies (Lafayette, CO). The knockdown of the transcripts was Cell Staining, Fluorescence Microscopy and Flow Cytometry To evaluate the extent of cell death, cultures were stained first with Hoechst 33258 (5 mg/ml, 15 min) washed and stained with propidium iodide (2.5 mg/ml, 15 min) and then visualized by PLoS ONE | www.plosone.org 11 March 2010 | Volume 5 | Issue 3 | e9516