Fv1000 asw 3

HMEC-1s were plated in 24-well cell-culture plates (Corning Inc., Corning, NY) with glass coverslips (Fisher Scientific, Rochester, NY) at a density of 5×10⁴ cells per well and allowed to attach overnight. After a 24 h treatment with control, SW480-derived EVs (1 µg/mL, 0.5 mL), or EGM-2, cells were fixed with 4% paraformaldehyde and incubated with rabbit anti-phospho-histone H3 (PH3) antibody (Upstate Biotechnology, Lake Placid, NY) followed by AlexaFluor488 goat anti-rabbit IgG antibody (Invitrogen). Nuclei were counterstained with Hoechst (Sigma-Aldrich). Images were visualized under an FV1000 Olympus confocal microscope (Olympus) and acquired using FV1000-ASW 3.0 software (Olympus). The percentage of PH3-positive cells was quantified by counting the cells with co-localized fluorescence signals.

SW480-derived EVs were labeled with DiI (Invitrogen) according to the manufacturer's instructions. Briefly, SW480-derived EVs (100 µg) were incubated with DiI (1 µM) for 15 min at 37°C and centrifuged at 100,000 g for 2 h at 4°C (Type 90 Ti fixed angle rotor with a k-factor of 126.4). The pellets, DiI-labeled EVs, were washed with phosphate buffered saline, and after ultracentrifugation at 100,000 g for 2 h at 4°C (Type 90 Ti fixed angle rotor with a k-factor of 126.4), were resuspended in phosphate buffered saline. HMEC-1s were pretreated without or MβCD (10 mM) for 0.5 h, and then incubated with DiI-labeled SW480-derived EVs (1 µg/mL, 0.5 mL) for 1 h. Cells were then fixed with 4% paraformaldehyde and nuclei were stained with Hoechst (Sigma-Aldrich). Images were visualized under an FV1000 Olympus confocal microscope (Olympus) and acquired using FV1000-ASW 3.0 software (Olympus).

HMEC-1s were plated in 24-well cell-culture plates (Corning Inc.) with glass coverslips (Fisher Scientific) at a density of 5×10⁴ cells per well and allowed to attach overnight. Cells were treated with SW480-derived EVs (1 µg/mL, 0.5 mL), fixed with 4% paraformaldehyde, and incubated with rabbit anti-Egr-1 antibody (Cell Signaling Technology, Hitchin, United Kingdom) followed by AlexaFluor488 goat anti-rabbit IgG antibody (Invitrogen). Nuclei were counterstained with Hoechst (Sigma-Aldrich). Images were visualized under an FV1000 Olympus confocal microscope (Olympus) and acquired using FV1000-ASW 3.0 software (Olympus). The percentage of Egr-1-positive cells was quantified by counting the cells with co-localized fluorescence signals.
To investigate the effect of signaling inhibitors (BioMol Research Laboratories, Plymouth Meeting, PA, USA) or methyl-β-cyclodextrin (MβCD; Sigma-Aldrich) on Egr-1 nuclear translocation, HMEC-1s were treated with ERK1/2 inhibitor (PD98059, 20 µM), p38 MAPK inhibitor (SB203580, 10 µM), JNK inhibitor (SP600125, 20 µM), Akt inhibitor (BML-257, 20 µM), or MβCD (10 mM) in the presence or absence of SW80-derived EVs (1 µg/mL).