Scale bar, 500?m. gene in SK-MEL-28 melanoma cells, which normally express high levels of CEACAM1, inhibited formation of a VM-like network, which was covered on reintroduction of CEACAM1. These results suggest that CEACAM1 differentially regulates formation of the VM-like network between malignancy cell types and implicate CEACAM1 as a novel therapeutic target in malignant malignancy. methods [, , ]. We established CEACAM1-overexpressing HT1080?cells and found that CEACAM1 suppresses VM-like network formation on Matrigel in HT1080?cells. Further, CEACAM1 negatively regulates A419259 cell proliferation, and migration. In contrast, knockout (KO) of CEACAM1 inhibits the development of VM-like networks and migration in SK-MEL-28?cells. Several functions for CEACAM1 have been reported in cancer malignancy; thus, our results indicate that CEACAM1 is usually a novel cell-dependent regulator A419259 of VM. Rabbit Polyclonal to SCN4B 2.?Materials and methods 2.1. Western blot For the western blot analysis, we used a slightly altered version of a previous method [, , , , ]. Cells were lysed in lysis buffer (50?mM Tris-HCl, pH 7.5, 150?mM NaCl, 0.1% (w/v) SDS, 1% (v/v) Triton X-100, 1% (w/v) sodium deoxycholate, and 1?mM PMSF) at 4?C with sonication. The lysates were centrifuged at 15,000for 10?min. Loading buffer (350?mM Tris-HCl, pH 6.8, 30% (w/v) glycerol, 0.012% (w/v) bromophenol blue, 6% (w/v) SDS, and 30% (v/v) 2-mercaptoethanol) was added to each lysate. After electrophoresis, proteins were transferred to polyvinylidene fluoride membranes and immunoblotted with anti-c-myc (9E10 hybridoma culture supernatant, Developmental Studies Hybridoma Lender, USA), anti–tubulin (#T5168, Merck KGaA, Germany), or anti-CEACAM1 (sc166453, Santa Cruz Biotechnology). Signals were detected by ECL using Western Lightning Plus-ECL (PerkinElmer, Inc., USA). 2.2. VM-like network formation assay HT1080 and SK-MEL-28?cells, suspended in culture medium, were seeded at 1.6??104?cells/well or 2.0??104?cells/well in 96-well plates that precoated with 40 L/well Matrigel (Corning Inc., USA) and cultured at 37?C. These cells were photographed at 4 and 6?h after seeding. We quantified VM-like network formation as explained . 2.3. Wound healing assay HT1080?cells and SK-MEL-28?cells were seeded at 2??105?cells/well in 24-well plates, cultured for 24?h A419259 to approximately 90% confluence, and wounded using a yellow pipette tip (WATSON, Japan). After being washed twice with PBS, the cells were cultured in serum-free DMEM for 12 and 24?h. Photographs were taken of 4 impartial areas, and the areas of migration were quantified using ImageJ 1.51 (National Institutes of Health, Bethesda, MD, USA) [27,28]. 2.4. Cell culture, plasmid construction; establishment of CEACAM1-overexpressing HT1080, CEACAM1-KO SK-MEL-28, and CEACAM1-rescued SK-MEL-28?cells; RNA extraction and semiquantitative PCR; cell proliferation assay; and statistical analysis Cell culture; plasmid construction; generation of CEACAM1-rescued SK-MEL-28, CEACAM1-overexpressing HT1080, and CEACAM1-KO SK-MEL-28?cells using the CRISPR/Cas9 system; RNA extraction and semiquantitative PCR; cell proliferation assay; and statistical analysis are explained in Supplementary Materials and Methods. 3.?Results 3.1. Expression of CEACAM1 in various cancer cell collection cultures The expression of CEACAM1 varies, depending on the type of malignancy [, , , , ]. We measured endogenous CEACAM1 levels by semi-quantitative RT-PCR and western blot in various malignancy cell lines. CEACAM1 expression was high in SK-MEL-28 and HepG2 cells but absent A419259 in HT1080 and PC-3?cells (Fig. 1A and B). These results indicate that CEACAM1 expression depends on the malignancy cell type. Open in a separate windows Fig. 1 Expression of endogenous mRNA in various human malignancy cell lines. (A) Total mRNA was isolated from HT1080, SK-MEL-28, HepG2, HCT116, PC-3, and A549?cells, and semi-quantitative RT-PCR was performed. (B) HT1080, SK-MEL-28, HepG2, HCT116, PC-3, and A549?cells were cultured, and the cell lysates were immunoblotted with the indicated antibodies. 3.2. Suppression of VM-like network formation in HT1080?cells by CEACAM1 overexpression Because we reported that HT1080?cells have the potential to form a VM-like network on Matrigel , we used HT1080?cells to measure CEACAM1 function with regard to VM. To determine whether CEACAM1 affects formation of a VM-like network, we established a CEACAM1-overexpressing HT1080?cell collection (Fig. 2A) and assessed its ability to form such a network on Matrigel. Network formation on Matrigel was significantly suppressed by the overexpression of CEACAM1 (Fig. 2B and C), suggesting that CEACAM1 inhibits VM-like network formation in HT1080?cells. Open in a separate windows Fig. 2 Effect of CEACAM1 overexpression on VM-like network formation in HT1080?cells. (A) Generation of CEACAM1-overexpressing HT1080?cells. Control (Luc) and CEACAM1-overexpressing HT1080?cells were cultured, and the cell lysates were immunoblotted with.