Fig. 4: PAX3-FOXO1 is sufficient to increase sensitivity to ATR inhibition in myoblast cells.
From: Therapeutic targeting of ATR in alveolar rhabdomyosarcoma
a Western immunoblot of PAX3-FOXO1 and RPA32 phosphorylation at T21 in C2C12 after doxycycline-induced expression of PAX3-FOXO1 (1000 ng/ml for 48 h) and treatment with AZD6738 (750 nM). Hydroxyurea (HU, 1 mM) was used as a control for replication stress. b Representative images of H2AX phosphorylation in C2C12 cells after ectopic expression of PAX3-FOXO1. c Quantification of H2AX phosphorylation in C2C12 cells after ectopic expression of PAX3-FOXO1 (P = 9.57 × 10−26). Dose-response curves of cell viability for C2C12 cells after ectopic expression of PAX3-FOXO1 and incubation with AZD6738 (d) or BAY 1895344 (e) (n = 3). f Quantification of TUNEL signal in C2C12 cells after induction of PAX3-FOXO1 with doxycycline and treatment with AZD6738 (n = 3; from top to bottom, P = 0.016; 1.84 × 10−4; 1.99 × 10−4). g Relative HR activity in C2C12 cells after induction of PAX3-FOXO1 with doxycycline (1000 ng/ml) and incubation with AZD6738 as measured using a GFP reconstitution assay based on repair of an SceI-mediated DNA lesion via homologous recombination (n = 3 biologically independent experiments; from top to bottom, P = 5.19 × 10−6; 3.67 × 10−7; 0.114). h Western immunoblot of PAX3-FOXO1 in Rh4 cells after doxycycline-induced (1000 ng/mL) expression of shRNAs targeting PAX3-FOXO1 compared to scrambled shRNA control for 48 h. i Dose-response curves for Rh4 after doxycycline-induced (1000 ng/mL) expression of shRNAs targeting PAX3-FOXO1 compared to scrambled shRNA control and treated with AZD6738 (n = 3). All statistical analyses correspond to two-sided student’s t-test; data presented as mean value ± error bars representing standard deviation.
