Fig. 1
Generation and functional analysis of human tankyrase knockout cell lines. a Immunoblot analysis of whole cell lysates from HEK293T knockout (KO) cell lines generated by CRISPR-Cas9 stained with antibody that detects tankyrases 1 and 2. b FISH analysis of mitotic HEK293T WT, 1KO, 2KO, or DKO mitotic cells with a 16p telo probe (green). HEK293T cells are trisomic for chromosome 16. DNA was stained with DAPI. Scale bar, 5 μm. c Quantification of the frequency of mitotic cells with cohered telomeres. Average of two independent experiments (n = 80 cells) ± SEM. *p ≤ 0.05, **p ≤ 0.01, students unpaired t-test. d Analysis of telomere restriction fragments isolated from HEK293T WT or TNKS DKO cells isolated from an early (E) time immediately following the CRISPR-Cas9 screen and at a late (L) time point following passaging for ~2 months, fractionated on agarose gel, denatured, and hybridized with a 32P-[CCCATT]3 probe. EtBr stain of total DNA is below. e Analysis of telomere restriction fragments isolated from HEK293T WT, 1KO, 2KO, or DKO cells isolated from population doubling (PD) 6, 45, or 90, fractionated on agarose gel, denatured and hybridized with a 32P-[CCCATT]3 probe. f Graphical representation of the mean telomere length determined using Telometric (Fox Chase Cancer Center). g Immunofluorescence analysis of HEK293T WT, 1KO, 2KO, or DKO cells following fixation with 2% paraformaldehyde and stained with β-tubulin antibody (green) and DAPI (blue). Scale bar, 5 μm. h Quantification of the frequency of cells with defective mitotic spindles. Average of two independent experiments (n = 66–71mitotic cells each) ± SEM. *p ≤ 0.05, **p ≤ 0.01, Student’s unpaired t-test
