Figure S2 from A Genome-Wide CRISPR Screen Identifies Genes Critical for Resistance to FLT3 Inhibitor AC220

Supplementary Figure 2. sgRNAs target SPRY3 specifically delete SPRY3 gene in AML cells and SPRY3 deletion confers the resistance to Crenolanib and SPRY1 or 2 knockouts are resistant to AC220. (A) Western analysis was performed with 6 SPRY3 knockout mutants AML cells and SPRY1-3, ERK1/2 and GSK3 protein level has been examined. (B) Western analysis was performed with 6 SPRY3 knockout mutants AML cells and SPRY4 protein level has been examined with GAPDH as the loading control. (C) Cell growth curve of Wild type and SPRY3 knockout AML cells following the treatment with Crenolanib. 0.4 X 106 cells were plated in 12-well plate and treated with indicated amounts of Crenolanib for 6 days and cell numbers were counted. (D) Top: Cell growth curve of SPRY1 and SPRY2 knockout AML cells following the treatment with AC220. 0.4 X 106 cells were plated in 12-well plate and treated with indicated amounts of AC220 for 6 days and cell numbers were counted. Bottom: T7EN1 assay analysis of specific sgRNA-mediated indels at SPRY1 or SPRY2 locus in MV4-11 cells. The MV4-11 cells were transduced with lentivirus carrying sgRNA targeted SPRY1 or SPRY2. The genomic DNA from the cells was extracted and PCR amplified to test efficiency of gene disruptions by T7 endonuclease I assay using a 1.5% agarose gel. The lower migrating bands in lanes indicate the disrupted gene alleles.