DNA content of LIP-expressing MDA-MB-468 cells.

<p>A, Representative cell cycle profiles of control uninfected: no virus (NV) (panel a), Ad-GFP (panel b), and Ad-LIP (panel c) MDA-MB-468 cells are shown. B, Quantification of the percent of cells with greater than 4n DNA content as measured by the area marked as R2 in the graphs is presented. Data shown represents the mean + SD of three separate experiments. *p-value <0.05 as determined by ANOVA followed by Friedman test.</p

Ultrastructure analysis of LIP-mediated cell engulfment in MDA-MB-468 cells.

<p>Panels a–c, ultrastructure of engulfing intermediates at 48 hrs post infection; scale bar is 2µm. 1 and 2 indicate higher magnification of cell contacts present in panel c. Arrows indicate formation of cell contacts. Scale bar is 100 nm and 500 nm for panel 1 and 2, respectively. Panels d and e show LIP-expressing cells that have enormous vacuoles; scale bar is 2 µm. Panels f–h illustrate an internalized cell at 72 hrs post infection; scale bar is 2 µm. Panel g is a higher magnification to show the nucleus of engulfing (host) cell and the various projections typical of actively phagocytic cells. Panel h depicts the engulfed cell inside of the vacuole of the host cell.</p

Quantification of LIP-mediated cell engulfment.

<p>A, Panel a shows the percent of Ad-LIP MDA-MB-468 cells that are double positive for GFP and CellTracker label. Panel b shows the percent of control Ad-GFP MDA-MB-468 cells that are double positive for GFP and CellTracker label. B, Quantification of the percent of GFP positive cells that have engulfed uninfected CellTracker labeled cells. Results are shown for 16 different experiments with a p-value of <0.0001 using paired t-test and Wilcoxon matched pairs test for statistical analysis. C, Quantification of the percent of GFP positive cells that have engulfed uninfected CellTracker labeled cells after treatment with 40 µM of the ROCK inhibitor, Y-27632. Results are shown for 5 different experiments with a p-value of <0.0001 using ANOVA followed by the Student-Newman-Keuls multiple comparisons test.</p

LIP-mediated cell engulfment is not dependent on adenoviral infection.

<p>MDA-MB-468 cells cotransfected with LIP and GFP expression vectors were stained with anti-beta-catenin antibody (red) and Hoechst nuclear stain (blue). GFP positive cells in panels a–f are LIP expressing cells. Panels a–f are fluorescent micrographs of separate, randomly chosen fields; cells were imaged 4–6 days post transfection.</p

Cell disintegration following exogenous expression of LIP.

<p>Microscopic examination of cell breakdown. Equal numbers of MDA-MB-468 cells were plated for adenoviral infection with either control Ad-GFP or Ad-LIP. Representative fluorescent photomicrographs of cells imaged at 72 hrs post infection are shown. Panel a represents Ad-GFP MDA-MB-468 cells. Panels b-i represent Ad-LIP MDA-MB-468 cells.</p

Cells Expressing the C/EBPbeta Isoform, LIP, Engulf Their Neighbors

<div><p>Descriptions of various processes that lead to cell-in-cell structures have been reported for decades. The exact molecular mechanism(s) of their formation and the physiological significance of cell-in-cell structures remain poorly understood. We had previously shown that an isoform of the CCAAT/enhancer-binding protein beta (C/EBPbeta) transcription factor, liver-enriched inhibitory protein (LIP), induces cell death in human breast cancer cells and stimulates autophagy. Here we describe a non-apoptotic cell death process where LIP mediates the engulfment of neighboring cells. We provide evidence of LIP-mediated engulfment via DNA profiling, fluorescent imaging and cell sorting studies, as well as ultrastructure analysis of LIP-expressing MDA-MB-468 breast cancer cells. Our work illustrates that expression of a specific transcription factor, LIP, can mediate cell engulfment.</p> </div

LIP-expressing MDA-MB-468 cells engulf neighboring cells.

<p>GFP positive LIP-expressing MDA-MB-468 cells were mixed with uninfected CellTracker orange labeled MDA-MB-468 cells. Hoechst dye was used to stain DNA. Cells were imaged at 36–48 hrs post-mix.</p

Mutation of Thr235 to alanine decreases sumoylation of C/EBPbeta1.

<p>a. Cos-7 cells were untransfected (lanes 1, 4, and 7), transfected with T7-C/EBPbeta1-pcDNA3.1 and HA-SUMO-2-pcDNA3 (lanes 2, 5 and 8), or transfected with T7-C/EBPbeta1T235A-pcDNA3.1 and HA-SUMO-2-pcDNA3 (lanes 3, 6, and 9). All samples were immunoprecipitated with T7 antibody beads. Immunoblot analysis was performed with the anti-phosphoThr235 C/EBPbeta antibody (left), anti-HA tag (middle), and anti-C/EBPbeta antibody (right). Arrows indicate sumoylated T7-C/EBPbeta1 and p52-T7-C/EBPbeta1. b. Immunoblot analysis using the anti-T7 tag antibody of cell lysates from Cos-7 (lane 1), Cos-7 cells transfected with T7-C/EBPbeta1-pcDNA3.1 and HA-SUMO-2-pcDNA3 (lane 2), and Cos-7 transfected with T7-C/EBPbeta1T235A-pcDNA3.1 and HA-SUMO-2-pcDNA3 (lane 3). Arrows indicate p52-T7-C/EBPbeta1 and sumoylated T7-C/EBPbeta1. The relative amount of protein in the parent T7-C/EBPbeta1 band and the 75 kDa sumoylated T7-C/EBPbeta1 band was measured using the LI-COR Odyssey system. It was determined that there is 3.25 times more sumoylated 75 kDa T7-C/EBPbeta1 as there is 75 kDa T7-C/EBPbeta1T235A. This was calculated relative to the p52-T7-C/EBPbeta1 and p52-T7-C/EBPbeta1T235A bands. This was repeated three times with a standard deviation of +/−0.26. (beta1 = C/EBPbeta1, su-beta1 = sumoylated C/EBPbeta1).</p

Sumoylation of C/EBPbeta1 in breast cancer cells.

<p>a. Cell lysates were prepared and were run on an 8% SDS-PAGE in the following order: lane 1 MCF10A, lane 2 MDA231, lane 3 MDA468, lane 4 BT-474, lane 5 SK-BR3, lane 6 MDA435 and lane 7 T47D. Immunoblot analysis was performed with a C/EBPbeta1-specific antibody raised to the first 21 amino acids unique to C/EBPbeta1 (Abcam 18F8). The bottom immunoblot was performed as a loading control for GAPDH. Bars indicate the mobility's of standard molecular weight markers, in kilo-Daltons (kDa), in all figures. b. MDA231 breast cancer cells were infected with T7-C/EBPbeta1-IRES-eGFP-LZRS three times and sorted by FACs using GFP as a marker. Immunoprecipitations of confluent 100 mm dishes were performed with uninfected MDA231s (lanes 1 and 3) or T7-C/EBPbeta1-MDA231 cells (lanes 2 and 4) using T7 antibody beads. The left is an immunoblot with an anti-SUMO-2/3 antibody and the immunoblot on the right is with an anti-C/EBPbeta antibody. Sumoylated C/EBPbeta1 is indicated and the parent p52 C/EBPbeta1 is indicated by the arrow. c. Immunoprecipitations of MDA468 cells were performed with protein A agarose beads cross-linked to a C/EBPbeta1-specific antibody (described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025205#pone.0025205-Eaton1" target="_blank">[7]</a>). Lanes 3, 6, and 9 are the immunoprecipitations whereas lanes 1, 4, and 7 are negative control beads only and lanes 2, 5 and 8 are negative control non-crosslinked beads incubated with MDA468 extract. The left immunoblot is performed with an anti-SUMO-2/3 antibody, the middle immunoblot with a C-terminal C/EBPbeta antibody (Abcam 47A1) and the right hand immunoblot with a C/EBPbeta1-specifc antibody (Abcam 18F8). Arrows indicate sumoylated C/EBPbeta1. (231 = MDA231, beta1 = C/EBPbeta1, su = sumoylated).</p

Sumoylated C/EBPbeta1 does not induce senescence.

<p>A. Equal cell numbers of the indicated cell lines were plated in 60 mm dishes and stained for senescence associated beta-galactosidase as per manufacturer's instructions (Cell Signaling Technology). Representative photomicrographs imaged with a light microscope are shown. B. Quantitative comparison of senescence associated beta-galactosidase positive cells. The experiment was repeated four times with equal cell numbers of the indicated cell lines ranging from 50,000–250,000 cells/60 mm dish. The density of plating did not affect the outcome. For each experiment the average number of beta-galactosidase positive (blue) cells in 10 fields was computed. Error bars indicated standard deviation of the mean. C. Whole cell extracts were prepared from the indicated WI-38hTert cells and analyzed by immunoblotting (bottom panel) or immunoprecipitated with T7 antibody beads (Novagen, upper panel) followed by immunoblotting. Immunoblot analysis was performed with N-terminal C/EBPbeta antibody developed in our lab and described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025205#pone.0025205-Eaton1" target="_blank">[7]</a>.</p