Additional file 1: of The Anatomy to Genomics (ATG) Start Genetics medical school initiative: incorporating exome sequencing data from cadavers used for Anatomy instruction into the first year curriculum

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Depletion of km23-1 inhibits cell migration and invasion of human CRC cells.

<p><b>A:</b> Transwell migration assays were performed as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066439#s2" target="_blank">Materials and methods</a>.” Briefly, HCT116 cells stably transduced with lentiviral vectors expressing either piLenti-NC siRNA-GFP or piLenti-km23-1 siRNA-GFP were seeded into the upper wells of the Costar Transwell System (8-µm pore size polycarbonate membrane, 6.5-mm diameter), and the cells on the lower surface of the well after 24 h were fixed in methanol and stained with DAPI. <b>Top</b>, representative images of the lower surface of the membrane are shown (100× magnification). <b>Bottom</b>, the number of migrating cells of both NC siRNA- and km23-1-siRNA-HCT116 stable transfectants were counted under a fluorescence microscope and statistically analyzed. *p<0.01 compared to NC siRNA. <b>B:</b> Matrigel invasion assays were performed with the indicated RKO stable cells clones (clones #1, 5) for 24 h using EGF (20 ng/ml) as the stimulus as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066439#s2" target="_blank">Materials and methods</a>.” Invaded cells were stained with 0.2% crystal violet. <b>Top</b>, representative images of the lower membrane surface from Matrigel are shown (100× magnification). <b>Bottom</b>, the number of invading cells for both NC siRNA and km23-1-siRNA HCT116 stable transfectants were counted under a light microscope and statistically analyzed. *p<0.01 compared to EV.</p

Depletion of km23-1 blocks constitutive ERK activation in human CRC cells.

<p><b>A.</b> EV, NC siRNA, and km23-1 siRNA stable transfectants (clones 1 and 5) were used to isolate RNA. RT-PCR was performed and products were analyzed as described in the “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066439#s2" target="_blank">Materials and Methods</a>.” The data plotted are the mean ± SE of three independent experiments. *p<0.01 compared to the NC siRNA. <b>B:</b> Western blotting of phospho- and total protein expression levels for ERK1/2 in RKO human CRC cells. <b>Bottom</b>, DIC protein was assessed as a loading control. Data are representative of three independent experiments. <b>C</b>: Western blotting of phospho- and total protein expression levels for ERK1/2 in HCT116 cells stably transduced with the lentiviral particles described in the “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066439#s2" target="_blank">Materials and Methods</a>.” Top, confirms knockdown of endogenous km23-1. <b>Bottom</b>, GAPDH protein was assessed as a loading control. Data are representative of three independent experiments. <b>D:</b> CBS cells were infected with either pilenti-NC siRNA-GFP or pilenti-km23-1 siRNA-GFP pools. 24 h after infection, Western blotting was performed using the indicated antibodies. <b>Top</b>, confirms knockdown of endogenous km23-1. <b>Bottom</b>, DIC protein was assessed as a loading control. Three independent experiments were performed and representative figures are shown.</p

Depletion of km23-1 in RKO human CRC cells inhibits transactivation of the proximal AP-1 site in the TGFβ1 promoter, TGFβ1 secretion, c- Fos-DNA binding to the TGFß1 promoter, and Elk-1 activation.

<p><b>A:</b> RKO stable transfectants were transiently transfected with phTG5-Lux and luciferase assays were performed as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066439#pone.0066439-Liu1" target="_blank">[20]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066439#pone.0066439-Pandey1" target="_blank">[40]</a>. Data are representative of three independent experiments. <b>B:</b> TGFβ1 concentrations in CM from RKO stable transfectants were measured by ELISAs as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066439#pone.0066439-Liu1" target="_blank">[20]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066439#pone.0066439-Pandey1" target="_blank">[40]</a>. *p<0.01 compared to the NC siRNA. <b>C:</b> ChIP assays were performed <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066439#pone.0066439-Liu1" target="_blank">[20]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066439#pone.0066439-Pandey1" target="_blank">[40]</a> using either the human TGFβ1 promoter region from −410 to −260 (upper panel) or the hTERT promoter region from −1734 to −1584 (lower panel) with the indicated antibodies. Input: Equal samples of chromatin DNA without prior immunoprecipitation. <b>D:</b> Western blotting of phospho- and total protein expression levels for Elk-1 in RKO human CRC cells. Three independent experiments were performed and representative figures are shown.</p

km23-1-siRNA blocks tumor growth of RKO cells <i>in vivo</i>.

<p><b>A:</b> NC-siRNA RKO and km23-1siRNA RKO clone #1 and #5 cells (5×10⁶) were inoculated subcutaneously behind the right anterior forelimb of nude mice (n = 5). Tumor volumes were calculated as (length×width²)/2. *p<0.05 compared to NC, ANOVA. <b>B:</b> At the termination of the study, ERK activation and km23-1 knockdown in RKO xenografts were confirmed by Western blotting using the indicated antibodies. DIC, loading control.</p

Depletion of km23-1 reduces Ezrin expression in human CRC cells.

<p><b>A:</b> The cell lysates used for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066439#pone-0066439-g001" target="_blank">Fig. 1D</a> were further subjected to Western blot analysis of Ezrin expression <b>(Top). Bottom</b>, DIC loading. <b>B:</b> HCT116 cells were infected with either pilenti-NC siRNA-GFP or the pilenti-km23-1 siRNA-GFP set. 24 h after infection, Western blotting was performed using the indicated antibodies. <b>Top</b>, confirms knockdown of endogenous km23-1. <b>Bottom</b>, DIC protein was assessed as a loading control. <b>C:</b> HCT116 stable pools that migrated through to the lower membrane in the Matrigel invasion assay were stained with an Ezrin Ab, followed by cy3-conjugated goat anti-rabbit IgG (red). DAPI staining permitted visualization of nuclei of individual cells (blue). GFP was used as a marker to designate cells transduced with siRNA (green). The cells were analyzed by an Olympus IX81 microscope at a magnification of 1000× with the appropriate filter sets.</p