Fig. 5

Generation and characterization of a cellular model of POLE deficiency in LN-229 glioblastoma cells. (a) Multicolor fluorescence in situ hybridization analysis of a metaphase chromosome preparation from an LN-229 glioblastoma cell. In the hyperdiploid karyotype, the long arm of chromosome 12 where POLE is localized (12q24.33), and the long arm of chromosome 19 where POLD1 is localized (19q13.33) are probably present in three copies. (b) Electropherograms of the sequence of the sgRNA on-target site in exon 2 of POLE. A clone with no mutational event at the sgRNA on-target site (clone 2, POLE WT) was selected as control. A single clone was devoid of WT POLE alleles and exclusively harbored the POLE:c.84_85del p.(V29Ffs*14) frameshift variant at the sgRNA on-target site (clone 17, POLE KO). The position of the deletion is indicated by an arrow. (c) Western blot analysis of LN-229 POLE WT and KO cell lysates using anti-POLE and anti-β-tubulin antibodies. No full-length POLE was detected in the POLE KO clone. (d) Representative flow cytometry plots and histograms of BrdU pulse-labeled and 7-AAD stained LN-229 POLE WT and KO cells at different time points after BrdU treatment. (e) Quantification of the mid-S phase BrdU-positive LN-229 POLE WT and KO cell subpopulations relative to the value 0 h after BrdU treatment showing a significant increase in POLE KO cells 10 h after BrdU compared to POLE WT cells suggesting delayed S phase progression. Given are mean and standard deviation of three independent experiments. (f) Representative image of a HPRT1 mutation assay using LN-229 POLE WT and KO cells treated with 6-TG for 5 days and stained with crystal violet. (g) Quantification of grayscale images of the HPRT1 mutation assay showing a significantly increased mean crystal violet staining intensity indicating resistance after treatment with 0.5 and 5 µM 6-TG in LN-229 POLE KO compared to WT cells suggesting a higher mutation rate. Given are mean and standard deviation of three independent experiments. 7-AAD, 7-aminoactinomycin D; BrdU, 5-bromo-2′-deoxyuridine; KO, knockout; PAM, protospacer adjacent motif; sgRNA, single guide RNA; 6-TG, 6-thioguanine; WT, wildtype. *, p ≤ 0.05; **, p ≤ 0.01; two-tailed Student’s t test