Figure 6.

Extracellular vesicles induce NfκB nuclear traslocation. (A) HVECs were treated with serum-free medium for 4 h (starvation), 10 ng/ml TNF-α for 30 min, with MM EVs pre-incubated with anti-CD63 antibody (MM + m anti-CD63) or with MM serum after serial ultracentrifugation steps (SN3 MM). Cells were fixed with 4% PFA, permeabilized with 0.2% Triton X-100, 2 mg/ml BSA, 1 mM NaN₃ in PBS, and stained with anti-NfκB antibody, followed by Alexa 488-conjugated anti-rabbit immunoglobulin (Ig) and DAPI. Scale bars, 5 μm. Coverslips were mounted using an anti-fade mounting medium (ProLong Gold-Invitrogen) on a glass slide. Fluorescent microscopy was performed on a ZEISS Axiovert 100 fluorescent microscope using the 63× Zeiss oil immersion objective. Single sections are shown for each condition. Images were processed with the use of Image pro-plus 4.5.1. (B) Nuclear NfκB fluorescence intensity was measured after different treatments (100 cells each experimental point) using ImageJ program. Significant differences were determined with Student’s t-test: *p < 0.05, **p < 0.01, ***p < 0.001. Values were shown as mean values ± SEM of at least 3 experiments. (C) WB analysis of cell extracts (homogenate, H, 25 μg) and nuclear extract (N, 50 μg) were performed with anti-NfκB antibody and with anti-Histone 3 antibody.