Fig 1: Screening protocol and targets validation.a Schematic representation of the screening protocol. 66 × 106 millions of cells were infected with the lentiviral particles containing the shRNA plasmids with a MOI of 0,1 for 2 days. For the selection of the expressing-shRNAs, cells were first grown in the presence of puromycin (2 µg/ml) for additional 3 days and subsequently treated with G5 (2.5 µM) for 60 h. Surviving cells were harvested to recover the genomic DNA. b Representation of the most enriched hits. Data are represented as fold enrichment respect to the median of all shRNAs. The enrichment is relative to the second most abundant shRNA. Only shRNA with a fold increase >3 are shown. c Hits that are in relationship with GSK3ß as up-stream regulators or downstream effectors. d U87MG cells were silenced for CAPN1 and PP2AC and, after 48 h from transfection, they were treated with G5 (2.5 µM) for 24 h. Cell death was calculated as percentage of cells positive to PI staining using cytofluorimetric analysis. Data are from three experiments; +SD. e Cellular lysates from the silenced cells were analyzed by immunoblot. Antibodies anti-CAPN1, anti-PP2AC, and anti-Actin (as loading control) were used as indicated. f U87MG and U87MG-BCLXL cells were silenced for GSK3ß. After 48 h they were treated with G5 (2.5 µM) for further 24 h. Cell death was calculated as percentage of cells positive to PI staining using cytofluorimetric analysis. Data are from three experiments; +SD. g qRT-PCR analysis of GSK3ß mRNA levels in silenced cells. Data were from three experiments; +SD. h Immunoblot analysis of GSK3ß levels in the indicated clones of U87MG cells, selected after CRISPR/Cas9 mediated knock-out. Actin was used as loading control. i Cytofluorimetric analysis were performed to measure the % of PI positivity in the different U87MG clones treated with G5 (10 µM) for 24 h. Data are presented as mean ± SD. n = 3.