Chemical compounds were purchased from Prestwick Chemicals (Illkirch, France) corresponding to 1200 approved drugs [U.S. Food and Drug Administration (FDA), European Medicines Agency (EMA), and other agencies] dissolved in DMSO at 10 mM. A second library of 2824 compounds was provided by the NCI chemical libraries as follows: diversity set III, 1596 compounds; mechanistic set, 879 compounds; approved oncology drugs set II, 114 agents; and natural products set II, 235 agents. All NCI stock compounds were received in DMSO at a concentration of 10 mM except for mechanistic set (at 1 mM) (in a 96-well plate format). All libraries were reformatted in-house in 384-well plates. BFA and nocodazole were purchased from Sigma-Aldrich and used as control molecules.

For compound screening, cells (5.0 × 103 per well) were seeded on black clear-bottom 384-well plates (ViewPlate-384 Black, PerkinElmer) in 40 μl of complete medium. The screen was performed at the similar early cell passages (±2) for both replicates. Twenty-four hours after cell seeding, compounds were transferred robotically to plates containing cells using TeMO (MCA 384) (TECAN) to a final concentration of 10 μM and 0.5% of DMSO. Controls were added to columns 1, 2, 23, and 24 of each plate. After 90 min of compound incubation, cells were treated with 40 μM biotin for 45 min (for TNF) or 120 min (CCR5) at 37°C. Compound screens were performed in two independent replicate experiments at the BioPhenics Screening Laboratory (Institut Curie).

Cells were processed immediately after biotin treatment for immunofluorescence. Briefly, cells were fixed with 3% paraformaldehyde for 15 min and quenched with 50 mM NH4Cl in phosphate-buffered saline (PBS) solution for 10 min. For cell surface labeling, cells were incubated with anti-mouse GFP (1:800, Roche, catalog no. 814 460 001) diluted in 1% bovine serum albumin blocking solution for 45 min. Cells were then washed with PBS and incubated for 1 hour with Cy3-conjugated anti-mouse (1:600; catalog no. 715-165-151, Jackson ImmunoResearch). Nuclei were counterstained with DAPI (Life Technologies) for 45 min.

Image acquisition was performed using an INCell 2200 automated high-content screening fluorescence microscope (GE Healthcare) at a ×20 magnification (Nikon 20×/0.45). Four randomly selected image fields were acquired per wavelength, well, and replicate experiment. Image analysis to identify cells presenting predominantly cell surface or intracellular CCR5 and/or TNF localization was performed for each replicate experiment using the Multi Target analysis application module in the INCell analyzer Workstation 3.7 software (GE Healthcare). Results were reported as mean values from four image fields per well.

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