Ma5 15886

For live cell invasion assay, the cells were stained with cell tracker red (C7025, Invitrogen, Carlsbad, CA, USA) and cell tracker green (C34552, Invitrogen, Carlsbad, CA, USA) in 5 µg/mL and incubated for 20 min at 37 °C. For immunostaining, Zonula occludens-1 (ZO-1) (40-2200), cluster of differentiation 31 (CD31) (MA3100), Ras homolog family member A (RhoA) (MA1-011), Ras-related C3 botulinum toxin substrate 1 (Rac1) (701942), hypoxia-inducible factor 1-alpha (HIF-1α) (MA1-516), and matrix metallopeptidase 9 (MMP-9) (MA5-15886) were purchased from Invitrogen and used following the manufacturer protocols. For nuclei staining, 4′,6-diamidino-2-phenylindole (DAPI) (D9542, Sigma-Aldrich, St. Louis, MO, USA) was used. The samples were rinsed with DPBS and fixed using 4% paraformaldehyde in DPBS for 10 min at room temperature (RT). After permeabilization using 0.1% Triton X-100 in DPBS for 15 min at RT, samples were blocked with 1% bovine serum albumin (BSA) (Sigma-Aldrich, St. Louis, MO, USA) in DPBS for 1 h at RT. Then, the samples were incubated with primary antibodies overnight at 4 °C. After washing with 0.1% BSA, the samples were incubated with secondary antibodies for 2 h at RT and nuclei were stained with 300 nM DAPI. A confocal laser scanning microscope (CLSM) (LSM 510-META, Zeiss, Oberkochen, Germany) was used for imaging and analyses.

Total protein was prepared from cells and tissues with the xTractor buffer (TaKaRa) and then quantified by the Dc-assay kit (Bio-Rad, Munich, Germany). Samples (30 μg) were analyzed by electrophoresis with Mini-Protean TGX gels (Bio-Rad), and the resulting gels were electroblotted to nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany). Proliferating cell nuclear antigen (PCNA; 13-3900; Invitrogen), pro-inactive caspase 3 (procaspase 3; ab32150; Abcam, Cambridge, UK), cleaved caspase 3 (c-caspase 3; ab49822; Abcam), matrix metalloproteinase 9 (MMP9; MA5-15886; Invitrogen), HOXB7 (40-2000; Invitrogen), and GAPDH (39-8600; Invitrogen) antibodies were selected as primary antibodies, and anti-mouse or anti-rabbit IgG conjugated with horseradish peroxidase (ab6789 or ab97051; Abcam) was used as the secondary antibody. Blots were detected with the enhanced chemiluminescence (Bio-Rad), and the intensities of the bands were analyzed with ImageJ software (National Institutes of Health, Bethesda, MD, USA).