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. Author manuscript; available in PMC: 2024 Jul 24.
Published in final edited form as: Science. 2024 Feb 15;383(6684):763–770. doi: 10.1126/science.adg3224

Fig. 4. Repression of telomerase by long-range resection, RPA, and ATR signaling.

Fig. 4.

(A) Immunoblots for the indicated proteins performed on RPE1-superT cells treated with CRISPR/Cas12 crRNA targeting EXO1 or a non-targeting control (cntrl) followed by shRNAs targeting DNA2 or a scramble shRNA control. Ku70, loading control; * non-specific band. (B) Quantification of relative neotelomere formation by TaqMan qPCR in cells in (A) normalized to cells treated with the control crRNA and scramble shRNA. (C) Immunoblot for RPA70 in RPE1-superT cells treated with a control (cntrl) siRNA or two siRNAs targeting RPA70. γ-tub, loading control. (D) Relative neotelomere formation based on qPCR in cells in (C) normalized to cells treated with control siRNA. (E) Relative neotelomere formation based on qPCR 48 h after AdCas9 infection of RPE1-superT cells treated with DMSO or the indicated inhibitors. (F) Relative neotelomere formation based on qPCR 48 h after AdCas9 infection of pLenti-sgTSTaqMan-TS RPE1 cells treated with DMSO or ATRi-2. (G) Immunoblots for the indicated proteins in RPE1-superT cells after CRISPR/Cas12a targeting. γ-tub, loading control; * non-specific band. (H) Relative neotelomere formation based on qPCR in cells in (G) normalized to cells treated with the control crRNA. (I) Immunoblots for the indicated proteins from RPE1-ST cells after targeting with ATRIP crRNA or a control (cntrl) crRNA. γ-tub, loading control. (J) Relative neotelomere formation based on qPCR in cells in (I). (K) Relative neotelomere formation based on qPCR in RPE1-superT cells treated with the indicated Chk1 inhibitors normalized to cells treated with DMSO. (L) Schematic illustrating the inhibitory effect of ATR on neotelomere formation at resected DSBs. (M) Schematic of the lentiviral vector to test neotelomere formation at I-SceI-induced DSBs, showing the primers for neotelomere formation detection, the I-SceI site, and the TaqMan probe. (N) Effect of ATRi-2 on neotelomere formation at I-SceI-induced DSBs. Mean ± SD of at least 3 biological replicates. ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, two-tailed ratio-paired t-test in (B), (D), (E), (F), (H), (J), (K), and (N). DNA-PKcsi, AZD-6748, 10 μM; ATMi, KU55933, 10 μM; ATRi-1, VE-821, 10 μM; ATRi-2, Gartisertib/M4344, 0.3 μM in (E) and (F) and 1 μM in (J) and (N); Chk1i-1, CHIR124, 0.25 μM; Chk1i-2, MK-8776, 1 μM; Chk1i-3, CCT245737, 1 μM.