Gli1 expression disrupts the ATR–Chk1 pathway. (A and C) HEK293 cells were transfected with empty vector (EV), EGFP-Gli1 expression vector, or EGFP-Gli1^ΔZFD expression vector and incubated for 20–24 h. Cells were left untreated (−) or exposed to 6 Gy IR and harvested 1 h later, and extracted proteins were immunoblotted as indicated. Asterisk indicates nonspecific antigen recognized by this antibody. (B) HEK293 cells transiently transfected with empty vector (−) or the EGFP-Gli1 expression vector (+) were treated with 6 Gy IR or 10 mM hydroxyurea. 1 h later the cells were harvested and extracted proteins were immunoblotted as indicated. Molecular mass markers (in kilodaltons) are indicated on the left side of immunoblots. (D) HEK293 transiently transfected with empty vector (−) or the EGFP-Gli1 expression vector (+) were treated with nothing (−) or 25 Gy IR and processed to separate unbound (Released) from chromatin-bound Rad9. Samples were then immunoblotted for Rad9. Molecular mass markers (in kilodaltons) are indicated on the left side of immunoblots. (E) HEK293 cells were transfected with empty vector (EV) and, as indicated, plasmids that encode EGFP, the EGFP-Gli1 expression vector, and SFB-Claspin. 24 h later, the cells were treated with nothing (−) or 6 Gy IR (+) and lysed 1 h later. SFB-Claspin was precipitated (IP) from cell lysates and the precipitates were washed and sequentially immunoblotted for Chk1 and S tag (to detect Claspin). Cell lysates were sequentially immunoblotted for Chk1 and EGFP (to show EGFP and EGFP-Claspin expression).

Gli1 expression increases genomic instability. (A) HEK293 cells were transiently transfected with empty vector (EV), the EGFP-Gli1 expression vector, or luciferase (Luc) siRNA or Chk1 siRNAs (insets above graph). Metaphases were scored for chromatid-type, chromosome-type, and other (dicentrics, rings, anaphase bridges, and pulverized chromosomes) aberrations. The graph shows the total number of aberrations counted in each sample. The breakdown (in percent) of each aberration type is shown in the data tabulated below the graph. Data points show mean of 20 cells ± SD. The experiment has been repeated three times with similar results. (B) Representative photomicrographs of samples quantitated in A. Bars, 5 μm. Arrows indicate aberrations.

DNA damage responses in wild-type and Ptc1^+/− mice. (A) Mice were exposed to 3 Gy IR and killed when they showed symptoms of increased intracranial pressure. Number of mice evaluated in each group is as follows: wild-type P3–4, n = 8; wild-type P5–6, n = 16; Ptc1^+/− P3–4, n = 19; and Ptc1^+/− P5–6, n = 22. The brain of each mouse was removed and inspected for the presence of tumor. All mice that died had a grossly apparent brain tumor in the cerebellum. (B and C) Wild-type and Ptc1^+/− P5–6 mice were untreated or irradiated. 1 h later, the cerebella were dissected and lysed. Each lane represents the cerebellum from a separate mouse of the indicated genotype. Extracted proteins were immunoblotted for P-Ser¹⁹⁸¹-ATM and P-Ser³¹⁷-Chk1. Membranes were reblotted with ATM and Chk1 antibodies, respectively. Molecular mass markers (in kilodaltons) are indicated on left sides of immunoblots.

Gli1 expression disrupts Chk1-dependent responses. (A) S-phase checkpoint assay in HEK293 cells stably transfected with empty vector (EV) or EGFP-Gli1 (inset). 1 h before irradiation, the cells were treated with vehicle or 3 mM caffeine (Caff). Total counts per minute for the nonirradiated samples in each set were 20,400 ± 800 (EV), 20,900 ± 600 (Gli1), 15,400 ± 900 (EV + Caff), and 15,400 ± 700 (Gli1 + Caff). Data points show mean of three samples ± SD from one representative experiment. The experiment has been repeated four times with similar results. (B) Clonogenic assay of wild-type (+/+) or Ptc1^+/− MEFs. MEFs (1,000 cells/well) were plated, irradiated, and incubated to allow colony formation. The plating efficiencies for wild-type and Ptc1^+/− MEFs were 23.4 and 19.9%, respectively. Data points show mean of three samples ± SD from one representative experiment. The experiment has been repeated two times with similar results. (C) HEK293 cells transiently transfected with empty vector (EV), EGFP-Gli1, or EGFP-Gli1-ΔZFD (inset) were plated (300 cells/well, 0–3 Gy; 1,000 cells/well, 5 Gy), exposed to IR, and allowed to form colonies. Data points show mean of three samples ± SD from one representative experiment. Plating efficiencies for EV-, Gli1-, and Gli1-ΔZFD–transfected cells were 22.3, 20.4, and 16.7%, respectively. The experiment has been repeated three times with similar results.