Figure 3
From: A genome-scale CRISPR/Cas9 knockout screening reveals SH3D21 as a sensitizer for gemcitabine
SH3D21 is a gemcitabine sensitizer. (a) Cell viability assay of Panc1 cells carrying sgRNA targeting SH3D21. Cell viability was assessed 72 h after start of the gemcitabine treatment. Control sgRNA data is the average of two control sgRNAs. Bars show the average of three experimental replicates ± SD. * and ** represent p-values ≤ 0.05 and ≤0.001, respectively. (Inset) SH3D21 knockout decreases the EC50 of gemcitabine in Panc1 cells, 41.1 nM in SH3D21-knockout cells vs 56.8 nM in control cells. (b) SURVEYOR assay of SH3D21 sgRNA. Genomic region containing the sgRNA target site was PCR-amplified, 800 bp. Control and sample amplicons were mixed and reannealed. The annealing product was digested by T7 endonuclease enzyme. Indel mutations of SH3D21 produce bands with the size of 480 and 320 bp. (c) si-SH3D21 increases sensitivity of Panc1 cells to gemcitabine. (d) Western blot of SH3D21 protein after treatment of Panc1 cells with the si-SH3D21. Panels are cropped images of the same blot stained with different antibodies. Full-length blots are presented in Supplementary Fig. S3. (e) Re-expression of SH3D21 in SH3D21-knockout cells rescued the effect of SH3D21 knockout. SH3D21-knockout cells were seeded and transfected with either SH3D21 expression vector or empty vector. After 24 hours the medium was change to the medium containing gemcitabine and cell viability was measured after 72 hours. (f) Western blot of SH3D21 re-expression in SH3D21-knockout cells.
