4 6 diamidino 2 phenylindole dapi

Ten thousand OVTOKO and OVISE cells, which overexpressed FLAG-MCM2-FL and 3×FLAG-MCM2-ΔN, were cultured on Falcon^® 4 Well Culture Slides (BD Falcon NJ, USA). Cells were fixed in 100% ethanol at −20° C for 20 min and then incubated with rabbit monoclonal anti-FLAG antibody (Sigma) at a 1:100 dilution in PBS for 1 h at room temperature. Then, they were stained with a TRITC-conjugated anti-rabbit antibody (Dako Cytomation, Glostrup, Denmark) at a 1:100 dilution for 20 min at room temperature. Slides were washed three times with PBS and mounted with mounting medium (Dako Cytomation) containing 4′,6-diamidino-2-phenylindole (DAPI, Abbott Molecular Inc., Des Plaines, IL, USA). Images were acquired using a FV1200 laser-scanning microscope (OLYMPUS, Tokyo, Japan) with a 1,000× objective.

Snap-frozen tissue sections derived from seven organotypic breast tumors or MCF-7 cells were fixed in 4% paraformaldehyde (PFA) (Sigma-Aldrich), and incubated with a primary antibody to phospho-histone H2AX (pSer¹³⁹) (γH2AX, 1:700, Sigma-Aldrich) for 16 h at 22°C. Anti-rabbit 647 or 548 Alexa Fluor-conjugated was used as a secondary antibody (Molecular Probes, Invitrogen), and nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Abbott Laboratories, Abbott Park, IL, USA). Slides were scored by fluorescence microscopy using an AxioImager Z1 microscope (Carl Zeiss, Göttingen, Germany) and photographed images were arranged with Photoshop. For each sample, 100 cells were analyzed and the H2AX phosphorylation index was calculated as a percentage of γH2AX-positive cells
[26 (link)].

The exosomes were isolated from the HGC-27 cells transfected miR-23a inhibitor, NC-inhibitor, miR-23a mimic or NC-mimic (exo-miR-23a inhibitor, exo-NC-inhibitor, exo-miR-23a mimic, or exo-NC-mimic). The PBS-suspended exosomes were mixed with carboxy fluorescein succinimidyl ester (CFSE) (C1031, Beyotime Institute of Biotechnology, Shanghai, China) at a volume ratio of 10:1, and incubated at 37°C for 10 min, which was then terminated with 100 μL stop buffer followed by incubation at 4°C for 30 min. After 3-min centrifugation at 14,000 rpm, the fluorescence-labeled exosomes resuspended in 200 μL PBS were then mixed with exosome-free medium. The exosomes were co-cultured with the 50–60% confluent HUVECs. HUVECs were incubated only with PBS as control. Meanwhile, exo-miR-23a mimic was co-cultured with HUVECs transfected with overexpressed (oe)-PTEN or oe-NC. After incubation in 24-well plates for 48 h, the nuclei of the HUVECs were stained with 4',6-diamidino-2-phenylindole (DAPI, Abbott, USA) and subsequently observed under a confocal microscope (FV10i, Olympus Optical Co., Ltd, Tokyo, Japan) to determine the internalization of exosomes derived from the GC cells. Finally, the expression of miR-23a was quantified by RT-qPCR.