HTS

For the automatic screening, made in collaboration with the TIGEM High Content Screening (HCS) Core, cells were plated in 384-well plates at a concentration of 6,000 cell/well in a total volume of 40 μL. The commercially available SELLECK kinase inhibitors library, composed of 273 kinase inhibitors, was kindly provided by the TIGEM HCS core.

Dual AAV2 (5 × 10⁴ GC/cells) and compounds were diluted separately in 5 μL/well of complete medium and then added to the cells.

Before the analysis, cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 7 min and stained with DAPI (1 mg/mL). Images were acquired and analyzed using the OPERA system and the Accapella-based Columbus software, using a script for the evaluation of number of total cells, eGFP-positive cells, and mean fluorescence intensity in eGFP-positive cells.

The screening was performed in triplicate, and Pearson's correlation coefficients were as follows: total number of cells replicate 1 versus 2 = 0.66, replicate 2 versus 3 = 0.71, replicate 1 versus 3 = 0.72; eGFP-positive cells/total cells replicate 1 versus 2 = 0.93, replicate 2 versus 3 = 0.91, replicate 1 versus 3 = 0.89; mean fluorescence intensity in eGFP-positive cells replicate 1 versus 2 = 0.86, replicate 2 versus 3 = 0.89, replicate 1 versus 3 = 0.83. Pearson's correlation values support the reproducibility of the screening replicates.⁴⁰