”type”:”entrez-protein”,”attrs”:”text”:”ODZ10117″,”term_id”:”1065476890″,”term_text”:”ODZ10117″ODZ10117 Reduces the Migration and Invasion of Glioblastoma Cells The migration and invasion of cancer cells into the bloodstream and surrounding tissues are critical steps in cancer metastasis, and the transcription of target genes associated with these processes is regulated by STAT3 in the tumor microenvironment [23]. well in 96-well plates and incubated in culture medium until 70C80% confluence. The cells were further incubated for 24 h with either vehicle alone or various concentrations of “type”:”entrez-protein”,”attrs”:”text”:”ODZ10117″,”term_id”:”1065476890″,”term_text”:”ODZ10117″ODZ10117. Cell viability was measured at 450 nm using microplate reader (Molecular Devices, Sunnyvale, USA) after being further incubated for 2C4 h at 37 C following the addition with EZ-CyTox Enhanced Cell Viability Assay Reagent (Daeil Lab Service, Seoul, Korea). 2.7. Immunofluorescence Staining Cells grown in lysine-coated 24-well plates were fixed for 45 min at room temperature in 3% paraformaldehyde in PBS and permeabilized for 10 min with 0.1% Triton X-100 in PBS. The plates were blocked for 20 min with STAT6 3% BSA in PBS and incubated with tyrosine phosphorylated STAT3 (pY705-STAT3) antibody at 4 C overnight. After washing with PBS, the Taltobulin dishes were incubated with fluorescein isothiocyanate (FITC)-conjugated secondary antibody at Taltobulin room temperature for Taltobulin 2 h. Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI, D8417, Sigma-Aldrich) and images were captured using a Zeiss Axiovert 200 inverted fluorescence microscope (Oberkochen, Germany) with an LSM 510 META system (ZEN 2011). pY705-STAT3 antibody was used at 1:200 dilution. 2.8. Tissue Staining and Immunohistochemistry Tissue samples were fixed with 4% paraformaldehyde in 0.5 M phosphate buffer and embedded in paraffin. The paraffin blocks were cut in 4-m-thick sections, mounted on glass slides, dewaxed, rehydrated with grade ethanol, and stained with hematoxylin and eosin (H&E, HT100132, Sigma Aldrich and S3309, Dako, Carpinteria, CA, USA). To perform immunohistochemical analysis, rehydrated slide sections were unmasked with 10 mM sodium citrate buffer, quenched Taltobulin endogenous peroxidase for 20 min in 3% hydrogen peroxide, blocked for 30 min in PBS containing 10% goat serum, and incubated at 4 C for overnight with appropriate primary antibodies with 1:100 dilution. The sections were incubated with biotinylated secondary antibody (anti-rabbit for BA-1000, anti-mouse for BA-9200 and anti-goat for BA-5000, Vector Labs, Burlingame, CA, USA) compatible with the primary antibody for 30 min, subsequently incubated with streptavidin-HRP (550946, BD Pharmingen, San Jose, CA, USA) for 40 min, and stained with 3,3-diaminobenzidine (“type”:”entrez-nucleotide”,”attrs”:”text”:”D22187″,”term_id”:”426322″,”term_text”:”D22187″D22187, Invitrogen). Digital images were obtained using the LAS Microscope Software (Leica Microsystems, Wetzlar, Germany). 2.9. Flow Cytometry Dissociated single cells of GSCs were washed with PBS and fixed with 4% paraformaldehyde at 4 C for 10 min in the dark. Fixed cells were washed twice in ice-cold FACS buffer (00-4222-26, eBioScience, Carlsbad, CA, USA) containing 3% BSA in PBS and incubated with phycoerythrin (PE)-conjugated CD133 Taltobulin antibody (130-113-108, 1:20 dilution, Miltenyi Biotec, Sunnyvale, CA, USA). After 1 h incubation at 4 C, the cells were washed twice with PBS and incubated with PE-conjugated avidin (554061, BD Pharmingen). To analyze cell cycle and apoptotic cell population, cells were fixed with 70% ice-cold ethanol, washed with PBS, incubated with RNase (50 g, 10109134001, Sigma Aldrich) at 37 C for 1 h, and stained with propidium iodide (PI, 20 g, 556463, BD Biosciences, San Jose, CA, USA) at 4 C in the dark. For Annexin V staining, Annexin V binding buffer (422201, BioLegend, San Diego, CA, USA) containing fluorescein isothiocyanate (FITC) conjugated with anti-Annexin V antibody (640906, 1:50 dilution, BioLegend) was used as manufacturers protocol. Stained cells were counted with flow cytometry using the BD LSRFortessaTM cell analyzer (BD Biosciences). 2.10. Wound Healing and Invasion Assays To conduct wound healing assay, cells were seeded into 12-well plates and then incubated over 90% confluence. The plate was scratched with pipette tips and washed with PBS. Cells were incubated for 24 h with fresh DMEM medium containing either vehicle alone or “type”:”entrez-protein”,”attrs”:”text”:”ODZ10117″,”term_id”:”1065476890″,”term_text”:”ODZ10117″ODZ10117. Digital images were obtained using.