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. 2017 Jan 13;45(5):2615–2628. doi: 10.1093/nar/gkw1295

Figure 4.

Figure 4.

TERC KO in H1299 cells results in engaging ALT pathway. (A) Overview of ALT cell line generation from telomerase positive cancer cells (H1299). H1299 TERC KO cells were generated by introducing with Cas9/TERC targeting guide RNA. H1299 TERC KO cells divided 40 population doublings before entering crisis. Single survival clone (H1299 ALT) was acquired. (B) DdTRAP analysis (bottom) and C-circle assay (upper) in H1299, H1299 TERC KO and H1299 ALT cells. (C) TRF analysis in H1299, H1299 TERC KO (pre-crisis, PD = 37) and H1299 ALT (post-crisis) cells. Digested DNA was run on 0.8% agarose gels. M indicates for the marker. (D) CsCl separation of leading and lagging strands in H1299 and H1299 ALT cells. a.u. represents arbitrary unit. (E) In-gel hybridization of leading and lagging strand telomeres in H1299 and H1299 ALT cells. (F) Quantification of G-overhang intensity in E. Relative ratio of native signal to denatured signal in leading or lagging strands was calculated (mean ± SEM; n = 2) ** indicates P < 0.01 and n.s. indicates non-significant (unpaired Student's t test).