{"id":3943,"date":"2019-06-13T08:07:37","date_gmt":"2019-06-13T08:07:37","guid":{"rendered":"http:\/\/biodigestor.net\/?p=3943"},"modified":"2019-06-13T08:07:37","modified_gmt":"2019-06-13T08:07:37","slug":"we-recently-reported-that-this-lectin-surfactant-protein-d-sp-d-suppresses","status":"publish","type":"post","link":"https:\/\/biodigestor.net\/?p=3943","title":{"rendered":"We recently reported that this lectin surfactant protein D (SP-D) suppresses"},"content":{"rendered":"

We recently reported that this lectin surfactant protein D (SP-D) suppresses epidermal growth factor receptor (EGFR) signaling by interfering with ligand binding to EGFR through an interaction between the carbohydrate-recognition domain name (CRD) of SP-D and A549 human lung adenocarcinoma cells were serum-starved overnight and incubated with 1 m gefitinib for 2 h at 37 C. the indicated antibodies. The display the densitometric evaluation, and data are presented as mean S.D. (same experiment as was performed using CHOK1 cells Vcam1<\/a> stably expressing human EGFR. display densitometric analyses, and data are presented as mean S.D. (test or Welch’s test was used for statistical comparisons. *, 0.05; **, 0.01 (compared with control). SP-A suppresses the proliferation, migration, and invasion of A549 cells Next, we examined the effects of SP-A around the proliferation of lung cancer cells. A549 cells were incubated with 10 g\/ml SP-A, and the cell proliferation was assayed after 24, 48, and 72 h. As shown in Fig. 2SP-A suppressed the proliferation of A549 cells. Dose dependence was also confirmed (Fig. 2A549 cells were plated in a 96-well plate (1 103 cells\/well), maintained in DMEM with 10% (v\/v) FCS, and incubated with 10 g\/ml SP-A at 37 C. The cell proliferation was assayed after 24, 48, and 72 h using the WST-1 reagent. The absorbance at 440 nm was measured on the plate reader. A549 cells were incubated with various concentrations of SP-A, and the cell proliferation was assayed after 72 h. The data shown are presented as mean S.D. (test or Welch’s test was used for statistical comparisons. *, 0.05; **, 0.01 (compared with the SGI-1776 kinase activity assay<\/a> control). A549 cells were incubated with the indicated concentrations of gefitinib with or without 20 g\/ml SP-A. Cell proliferation was assessed after 48 h using the WST-1 reagent. The data shown are presented as mean S.D. (test or Welch’s test was used for statistical comparisons. *, 0.05; **, 0.01 (compared with the control). A549 cells were seeded into the upper insert of a transwell double chamber in DMEM with 0.1% (v\/v) BSA and EGF (10 ng\/ml) with or without SP-A (10 g\/ml). DMEM with 10% (v\/v) FCS was added to the bottom wells as a chemoattractant. A control insert was used for migration assay (test or Welch’s test was used for statistical comparisons. *, 0.05; **, 0.01. A549 cells were seeded into the upper insert of a transwell double chamber using DMEM with 0.1% (v\/v) BSA and EGF (10 ng\/ml), with or without SP-A (20 g\/ml) or gefitinib (10 m). DMEM with 10% (v\/v) FCS was then added to the bottom wells as a chemoattractant. A control insert was used for the migration assay (test or Welch’s test was used for statistical comparisons. *, 0.05; **, 0.01. A549 cells were applied into each well of ibidi chambers. After incubation for 24 h, the culture inserts were removed, and the dishes were filled with a serum-free medium. EGF (100 ng\/ml) and SP-A (20 g\/ml) were added to the medium, and the cells were incubated for 24 h. The migrated cells were measured under a microscope. The data shown are the mean S.D. (test or Welch correction was used for statistical comparisons. *, 0.05; **, 0.01 (compared with EGF-treated control cells). We then SGI-1776 kinase activity assay evaluated the effects of SP-A around the migration and invasion of A549 cells. When SP-A was added, the number of EGF-induced migration and invasion cells was significantly decreased (Fig. 2dose-dependent suppression of EGF binding by SP-A. Binding of EGF to the cells was evaluated SGI-1776 kinase activity assay using a -counter as described under Experimental procedures. The data are expressed as relative values with the binding in the absence of SP-A being 100%. Experiments were performed in duplicate and were repeated three times. The data are representative of three impartial experiments. Open in a separate window Physique 4. SP-A does not influence cell-surface expression of EGFR in A549 cells. A549 cells were serum-starved overnight. The next day, cells were incubated with 20 g\/ml SP-A for 2 h, washed, and incubated with 0.5 mg\/ml Sulfo-NHS-LC-biotin for 30 min at 4 C. Whole-cell lysates were immunoprecipitated with the monoclonal anti-EGFR antibody (clone Ab-11) or control IgG. Samples were separated by SDS-PAGE, transferred onto PVDF membranes, and probed with HRP-conjugated SGI-1776 kinase activity assay streptavidin (and whole-cell lysate of A549 cells was immunoprecipitated (and experimental paradigm described in was performed in H441 cells (and sEGFR was produced in Flp-In CHOK1 cells and purified as described under Experimental procedures. 0.5 g of proteins with or without PNGase F treatment were subjected to SDS-PAGE, which was followed by Coomassie Brilliant Blue R-250 staining (indicated concentrations of SP-A were incubated with sEGFR (100 ng\/well).<\/p>\n","protected":false},"excerpt":{"rendered":"

We recently reported that this lectin surfactant protein D (SP-D) suppresses epidermal growth factor receptor (EGFR) signaling by interfering with ligand binding to EGFR through an interaction between the carbohydrate-recognition domain name (CRD) of SP-D and A549 human lung adenocarcinoma cells were serum-starved overnight and incubated with 1 m gefitinib for 2 h at 37… Continue reading We recently reported that this lectin surfactant protein D (SP-D) suppresses<\/span><\/a><\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[47],"tags":[3525,3524],"class_list":["post-3943","post","type-post","status-publish","format-standard","hentry","category-adenosine-a2b-receptors","tag-sgi-1776-kinase-activity-assay","tag-vcam1","entry"],"_links":{"self":[{"href":"https:\/\/biodigestor.net\/index.php?rest_route=\/wp\/v2\/posts\/3943"}],"collection":[{"href":"https:\/\/biodigestor.net\/index.php?rest_route=\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/biodigestor.net\/index.php?rest_route=\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/biodigestor.net\/index.php?rest_route=\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/biodigestor.net\/index.php?rest_route=%2Fwp%2Fv2%2Fcomments&post=3943"}],"version-history":[{"count":1,"href":"https:\/\/biodigestor.net\/index.php?rest_route=\/wp\/v2\/posts\/3943\/revisions"}],"predecessor-version":[{"id":3944,"href":"https:\/\/biodigestor.net\/index.php?rest_route=\/wp\/v2\/posts\/3943\/revisions\/3944"}],"wp:attachment":[{"href":"https:\/\/biodigestor.net\/index.php?rest_route=%2Fwp%2Fv2%2Fmedia&parent=3943"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/biodigestor.net\/index.php?rest_route=%2Fwp%2Fv2%2Fcategories&post=3943"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/biodigestor.net\/index.php?rest_route=%2Fwp%2Fv2%2Ftags&post=3943"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}