GoKawiilCancer dependency map analysis
Genome-wide gene dependency scores were obtained from the Cancer Dependency Map (DepMap) 2024 Q2 CRISPRGeneEffect.csv public data release47.
Cell line generation, exogenous expression, siRNA and cell viability assays
Cell lines were grown in RPMI 1640 medium supplemented with 10% FCS with the exception of HEK293T cells, which were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% FCS; CALU1 cells, which were grown in McCoy’s 5A, 10% FBS, 2 mM l-glutamine; and RERF-LC-AI cells, which were grown in MEM, 10% FBS, 2 mM l-glutamine. Cell line identity was verified by high-throughput single-nucleotide polymorphism (SNP) genotyping using Illumina Golden Gate multiplexed assays. SNP profiles were compared to SNP calls from internal and external databases to determine or confirm ancestry. All cell lines tested negative for mycoplasma contamination before storage/use at our institute.
siRNA KD experiments were performed using ON-TARGET-plus SMARTpools (siFBXO42 L-022191-00-0010, siCCDC6 L-010551-00-0010) or single SiGenome (siFBXO42 D-022191-02-0005) purchased from Horizon Discovery and were reverse transfected using Lipofectamine RNAiMax (Invitrogen) for 72 h according to the manufacturer’s instructions.
All constructs were generated through gene synthesis at Genscript with codon optimization for mammalian cell expression. Flag, HA or His tags were cloned at the N terminus of FBXO42 or CCDC6. FBXO42 deletion mutant amino acid boundaries were as follows: FBXO42(ΔF-box) (100–717), FBXO42(ΔKelch) (1–100), FBXO42(Δ1BC) (140–146), FBXO42(Δ)2BC (192–202) and FBXO42(ΔC-Ter) (2–295).
Generation of stable cell lines for expression of Flag–FBXO42 WT or mutants, sgRNAs, DOX-inducible CRISPR–cas9 or Flag–FBXO42 was carried out by the piggybac transposon system. Transfections were performed using Lipofectamine 3000 (Thermo Fisher Scientific) and cells selected for resistance to blasticidin (DOX-inducible expression) or puromycin (constitutive expression).
To assess clonogenic capacity, cells were seeded in 6- or 12-well plates at low confluency for 7 to 15 days. Cells were then fixed by 4% PFA and stained using 0.005% Crystal Violet.
Live-cell imaging was performed in a 96-well-plate format (×10 magnification) using the Incucyte Live-Cell Analysis System (Sartorius).
Cell lysates, IP, immunodepletion and WB
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