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The number of secreted sEVs and the protein levels of CD63 in TAMR-MCF-7 cells were decreased by the P2X7 antagonist, showing that P2X7 influences the production of sEV

The number of secreted sEVs and the protein levels of CD63 in TAMR-MCF-7 cells were decreased by the P2X7 antagonist, showing that P2X7 influences the production of sEV. production of sEV. Our results suggest that inhibiting the P2X7 could be considered for metastasis prevention in TAM-resistant cancer patients. and and for 15?min), the supernatant were applied for immunoblottings. Enhanced chemiluminescence (ECL) system reagent (EMD Milipore, Billerica, MA) was used for band detection. Intracellular calcium determination The intracellular calcium contents were measured using a FlexStation scanning fluorimeter with an integrated fluid transfer workstation (Molecular Devices, Sunnyvale, CA). 5??104 cells were seeded in 96-well plates and cultured overnight. Then, the final volume was adjusted to 200?L using the assay buffer of FLIPR Ca2+ assay kit (Molecular Devices), followed by incubation at 37?C for 1?h. Using the FlexStation, the intracellular calcium concentration was measured after setting the excitation, emission, and cut-off at 485, 525 and Rotundine 515?nm at 2.5-second intervals for 3?min. Total RNA isolation and reverse-transcriptase-polymerase chain reaction (RT-PCR) analysis Total RNA was extracted from MCF-7 and TAMR-MCF-7 cells using TRIzol reagent (Life Technologies, Grand Island, NY) according to the manufacturers protocol. 1?g total RNA was reverse-transcribed using Maxime RT-PreMix Kit (Intron biotechnology, Gyeonggi-do, Korea) to synthesize cDNA, and the obtained cDNA was amplified by PCR using a Maxime PCR PreMix Kit (Intron biotechnology, Gyeonggi-do, Korea) and electrophoresed on 2% agarose gel. Detailed primer sequences used in the experiments are provided as Supplementary Information (Supplementary Table?S1). Quantitative polymerase chain reaction was carried out using SYBR green (biorad, San Francisco, CA) incorporation with gene-specific primers. The forward primer for P2X7 was: 5- TATGAGACGAACAAAGTCACTCG-3 and the reverse one was: 5- GCAAAGCAAACGTAGGAAAAGAT-3. The primers for 18?S were: forward: 5- CCATCCAATCGGTAGTAGCG-3; reverse: 5- GTAACCCGTTGAACCCCATT-3. Relative gene expression was calculated by Ct analysis relative to 18?S. Real-time monitoring of cell proliferation 5??104 cells were seeded in 96 well-plate and the phase percentage of cells were scanned every Rotundine 4?h by using the IncuCyte ZOOMTM Live Cell Anaylsis System (Essen Bioscience, Ann Arbor, MI). Immunocytochemistry Coverglass sterilized with 70% ethanol was placed on a 24-well plate and 1??105 cells were seeded and the attached cells were incubated with vehicle or compound for 24?h. After washing twice with PBS, the cells were fixed with 4% paraformaldehyde for 20?min at room temperature and incubated with 0.1% Triton X-100 for 15?min. After washing three times with PBS, the fixed and permeable Cd200 cells were incubated with 10% horse serum for 1?h, and the cells were exposed to the primary antibody at 4?C overnight and subsequently fluorophore-conjugated secondary antibody. Then, the coverglass Rotundine was mounted on a slide glass and fixed with mounting gel, and the fluorecence images were obtained using iRiS ? Rotundine Digital Cell Imaging System (Logos Biosystems, Gyeonggi-do, Korea). Transwell migration and wound healing assays Cell migration assay was quantified by both trans-well migration and wound healing assays. For transwell migration assay, MCF-7 and TAMR-MCF-7 cells were seeded in the upper chamber of the trans-well plate and the lower chamber was filled with 10% FBS-containing media. The cells were incubated at 37?C for 18?h and then fixed with 4% formalin and methanol, and subsequently stained with hematoxylin and eosin. With 40 magnification, migrated cells to the lower filter side were analyzed. Also some parts of transwell migration assay were performed using Incucyte ZoomTM Live Cell Analysis System (Essen bioscience) with using Incucyte clearview chemotaxis plate. For wound-healing assay, cells were seeded at 4??104 cells/well in 96-well ImageLock plate (Essen bioscience) and the cells were incubated until 100% confluency. 96-well WoundMaker (Essen bioscience) was used to make Rotundine consistent scratch. After adding 100?L culture media to 96-well plate, wound scratch was photographed every 4?h using Incucyte Zoom and the change pattern was analyzed. P2X7 siRNA and transfection To improve the gene silence efficacy, two different P2X7 receptor siRNA (Bioneer, Daejeon, Korea) were combined: (1) sense:5-CUCUUGAGGAGCGCCGAAA-3; antisense:5-UUUCGGCGCUCCUCAAGAG-3 (2) sense: 5-CAGUGAAUGAGUACUACUA\3; antisense: 5-UAGUAGUACUCAUUCACUG\3. The scrambled siRNA was used as the control. 200?pmol P2X7 receptor siRNA or scrambled siRNA, was transfected using Lipofectamine 2000 (Life Technologies, Carlsbad, Ca) according to the manufacturers protocol. Spleen-liver metastasis and.