Schematic outline of genome‐scale CRISPR‐Cas9 screen for genes whose KO suppresses MMC hypersensitivity of SCAI KO cells. LD80, 80% lethal dose; NGS, next‐generation sequencing.
MaGECK analysis of sgRNA enrichment in CRISPR screen in (A) following MMC treatment (n = 3 technical replicates; false discovery rate (FDR) of 0.2 indicated by dotted line). FA genes are highlighted in blue.
GO term analysis of significantly enriched genes (FDR < 0.2) in CRISPR‐Cas9 screen in (A), using Reactome pathways from PANTHER16.0.
Immunoblot analysis of FANCA siRNA knockdown efficiency in U2OS cells.
Clonogenic survival of U2OS and U2OS/SCAI KO cells transfected with non‐targeting control (CTRL) or FANCA siRNAs and subjected to indicated doses of MMC for 24 h (mean ± SEM; n = 3 independent experiments).
U2OS and U2OS/SCAI KO cells transfected with indicated siRNAs for 48 h were treated or not with MMC (90 nM) for 1 h, fixed 24 h later, and co‐stained with RPA2 antibody and DAPI. RPA2 foci were quantified by QIBC (≥ 3,000 cells analyzed per condition; mean ± SD; n = 3 independent experiments; *P < 0.05; ns, not significant, two‐tailed paired t‐test).
As in (F), except that cells were stained with γH2AX antibody (≥ 3,000 cells analyzed per condition; mean ± SD; n = 3 independent experiments; **P < 0.01; ns, not significant, two‐tailed paired t‐test).
Immunoblot analysis of the indicated U2OS cell lines.
Clonogenic survival of U2OS and U2OS/FANCA KO cells subjected to indicated doses of MMC for 24 h (mean ± SEM; n = 3 independent experiments).
Clonogenic survival of U2OS, U2OS/SCAI KO, U2OS/FANCA KO, and U2OS/FANCA+SCAI double KO (DKO) cells subjected to indicated doses of MMC for 24 h (mean ± SEM; n = 3 independent experiments).
Immunoblot analysis of U2OS, U2OS/SCAI KO, U2OS/FANCA KO, and U2OS/FANCA SCAI DKO cell lines exposed or not to MMC as indicated.
