FIGURE 3 from Lung Fibroblasts Take up Breast Cancer Cell-derived Extracellular Vesicles Partially Through MEK2-dependent Macropinocytosis

Abstract

<p>Screening of a targeted library of oncology drugs by high-content microscopy identifies compounds suppressing EV uptake. <b>A,</b> Cells were pretreated with drugs (200 nmol/L final concentration) for 24 hours and then either assayed for viability or incubated with 10 µg/mL EV for 6 hours and assayed for EV uptake. Dynasore (80 µmol/L) was also included as a control treatment. For both MTS assay and EV uptake assay, <i>n</i> = 2 wells per drug were tested. For EV uptake assay, 5 images per well were captured and analyzed. Each dot represents the mean value of two replicates. Drugs resulting in >75% cell viability and <75% EV uptake efficiency were indicated in red. <b>B,</b> Representative images of untreated cells and cells treated with selected drugs. Images were captured at 10 ×. Green, CFSE; yellow, DiI; blue, Hoechst 33342. Scale bar, 100 µm. <b>C,</b> Dose-dependent analysis of the effect of Trametinib and Copanlisib. Experiments were carried out as in A using indicated concentrations of the drug. Data are represented as mean ± SD (<i>n</i> = 3 wells per group; 5 images per well). The best-fit IC<sub>50</sub> of each drug is indicated. <b>D,</b> Validation of the effect of Trametinib and Copanlisib by flow cytometry. Experiments were carried out as in A but scaled up to a 6-well plate format. Data are represented as mean ± SD (<i>n</i> = 3 wells). ***, <i>P</i> < 0.001.</p>