Survey of Servers.

1<p><a href="http://fasta.bioch.virginia.edu/fasta_www2/fasta_www.cgi" target="_blank">http://fasta.bioch.virginia.edu/fasta_www2/fasta_www.cgi</a>,</p>2<p><a href="http://web.expasy.org/cgi-bin/protscale/protscale.pl" target="_blank">http://web.expasy.org/cgi-bin/protscale/protscale.pl</a>,</p>3<p><a href="http://bp.nuap.nagoya-u.ac.jp/sosui/sosui_submit.html" target="_blank">http://bp.nuap.nagoya-u.ac.jp/sosui/sosui_submit.html</a>,</p>4<p><a href="http://www.cbs.dtu.dk/services/TMHMM/" target="_blank">http://www.cbs.dtu.dk/services/TMHMM/</a>,</p>5<p><a href="https://www.predictprotein.org/" target="_blank">https://www.predictprotein.org/</a>,</p>6<p><a href="http://www.enzim.hu/hmmtop/html/submit.html" target="_blank">http://www.enzim.hu/hmmtop/html/submit.html</a>,</p>7<p><a href="http://www.sbc.su.se/~miklos/DAS/" target="_blank">http://www.sbc.su.se/~miklos/DAS/</a>,</p>8<p><a href="http://www.ch.embnet.org/software/TMPRED_form.html" target="_blank">http://www.ch.embnet.org/software/TMPRED_form.html</a>,</p>9<p><a href="http://distill.ucd.ie/paleale/" target="_blank">http://distill.ucd.ie/paleale/</a>,</p>10<p><a href="http://octopus.cbr.su.se" target="_blank">http://octopus.cbr.su.se</a>,</p>11<p><a href="http://pipe.scs.fsu.edu/wesa.html" target="_blank">http://pipe.scs.fsu.edu/wesa.html</a>,</p>12<p><a href="http://bioinf.cs.ucl.ac.uk/psipred/" target="_blank">http://bioinf.cs.ucl.ac.uk/psipred/</a>.</p

Size Distribution of Soft Agar Colonies.

<p>Image ProPlus software was used to calculate the average area of soft agar colonies. The results represent three separate soft agar growth experiments. The columns marked with an asterisk indicate sublines that were either significantly smaller or larger than those formed by cells transfected with wild type (WT) CEACAM1-4S (p<0.05).</p

Rates of Cell Proliferation of CEACAM1-4S Transfectants.

<p>The bars show the fold-increase in cells at 96 hours after plating. The columns marked with an asterisk were sublines showing an increase in cell number between 0 and 96 hours that according to P values (p<0.05), was significantly higher or lower than 253T-NT cells transfected with wild type (WT) CEACAM1-4S. In general, the number of 253T-NT cells expressing G to L mutants increased at a slower rate and Y to V mutants at a higher rate than cells expressing wild type CEACAM1-4S. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029606#s3" target="_blank">Results</a> shown represent four separate assays.</p

CEACAM1-4s Transmembrane Domain.

<p>CEACAM1-4s Transmembrane Domain.</p

Cell Surface Expression of CEACAM1-4S.

<p>253T-NT cultures stably transfected with wild type or mutated forms of CEACAM1-4S were labeled by indirect immunofluorescence with MAb 9.2, a monoclonal antibody specific for CEACAM1. Cell nuclei were stained with propidium iodide. Confocal digital images constructed from 7–15 optical sections for each labeled subline are shown in panels A–L. (<b>A</b>) wild type CEACAM1-4S; (<b>B</b>) G424L mutant; (<b>C</b>) G432L mutant; (<b>D</b>) G424L and G432L double mutant; (<b>E</b>) Empty vector; (<b>F</b>) Y445V mutant; (<b>G</b>) Y448V mutant; (<b>H</b>) Y445V and Y448V double mutant; (<b>I</b>) G424L, G432L, Y445V, Y448V quadruple mutant; (<b>J</b>) untransfected 253T-NT cells; (<b>K</b>) G424L, G432L and Y445V triple mutant; (<b>L</b>) G424L, G432L and Y448V triple mutant. Scale bar represents 20 mm.</p

Tumorigenicity of 253T-NT Transfected with WT and Mutated CEACAM1-4S.

<p>Three nude mice were injected in the front flanks with each subline of 253T-NT (6 injection sites for each cell line). At three weeks after injection, tumor nodules were harvested and weighed. Columns marked with an asterisk indicate cell lines that according to P values (p<0.05) were significantly larger (Y448V and the quadruple mutant) or smaller (G424L) than 253T-NT cells expressing wild type CEACAM1-4S. Although the effects of mutations were not as clear-cut as those observed for growth in soft agar, the G424L mutation compromised and the Y448V or the quadruple mutation enhanced the ability of CEACAM1-4S to produce tumors in nude mice.</p

Anchorage Independent Growth in Soft Agar.

<p>(<b>A–I</b>) show the appearance of colonies formed by the various CEACAM1-4S sublines after 3 weeks in soft agar: (<b>A</b>) G424L; (<b>B</b>) G432L; (<b>C</b>) G424L and G432L; (<b>D</b>) Y445V; (<b>E</b>) Y448V; (<b>F</b>) Y445V and Y448V; (<b>G</b>) G424L, G432L and Y445V; (<b>H</b>) G424L, G432L and Y448V; (<b>I</b>) G424L, G432L, Y445V and Y448V.</p

Immunoblot Analysis Shows that Wild Type and Mutant CEACAM1-4S Constructs Have Identical Molecular Mass.

<p>Protein lysates prepared from wild type cells and each of the 253T-NT transfected cell lines were resolved on 7.5% SDS-polyacylamide gels, transferred onto nitrocellulose and labeled with MAb 9.2 specific to CEACAM1. Analysis of expressed wild type and mutant CEACAM1-4S protein shows that reactive band corresponding to CEACAM1-4S had the same apparent molecular mass (105 kDa).</p

Mobility of G to L Mutants of CEACAM1-4S Resolved by BN-PAGE.

<p>Protein lysates were prepared and resolved by BN-PAGE as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029606#s2" target="_blank">materials and methods</a>. Proteins were transferred onto PVDF membranes and probed with monoclonal antibody 9.2. When separated on native gels, wild type CEACAM1-4S and the single G mutants migrated with an apparent molecular mass that was approximately 100 kDa higher than the double glycine mutant.</p