Fig. 4

CRISPR library screening identifies PTPN11 as a critical gene for elaiophylin-induced paraptosis. a A diagram for the genome-wide CRISPR/Cas9 knockout library screening. b Elaiophylin selection identified potential target genes. The top 10 positive selected genes were ranked by p value. c Top: SKOV3 cells were exposed for 24 h to DMSO or 0.5 µM elaiophylin and subject to CETSA. Bottom: the intensity of the SHP2 bands or PTPRM bands was quantified to exhibit the binding affinity of elaiophylin to SHP2 or PTPRM in SKOV3 cells. d SPR analysis showing direct interaction between elaiophylin and human recombinant SHP2 protein at the molecular level. e Pattern diagram of SHP2/SOS1/MAPK pathway. f Assessment of indicated protein levels using western blotting in SKOV3 cells exposed to 0.5 µM elaiophylin. g Left panel: representative light microscopy images of SKOV3 and OVCAR8 cells exposed to 0.5 µM elaiophylin for 9 h after PTPN11 knockdown. Arrows indicate cytoplasmic vacuoles. Scale bar: 50 µm. Right panel: proportion of cells displaying vacuoles was scored by visually examining at least 100 cells. Data are mean ± SD of three independent experiments (Two-tailed unpaired Student’s t test, *p < 0.05, ***p < 0.001). h Assessment of indicated protein levels using western blotting in SKOV3 cells exposed to 0.5 µM elaiophylin for 9 h after PTPN11 knockdown. i The viability of SKOV3 and OVCAR8 cells exposed to 0.5 µM elaiophylin for 36 h after PTPN11 knockdown. Data are mean ± SD of three independent experiments (Two-tailed unpaired Student’s t test, **p < 0.01, ***p < 0.001)