Figure 1. ATRX-deficient human glioma cells demonstrate inappropriate cell cycling after treatment with irradiation.

(A) Western blot of U251, SF188 and UW479 isogenic ARTX^KO cells illustrating ATRX reduction. Immunoblots of KNS42 and SJ-GBM2 are also included (right two columns) illustrating ATRX reduction in ATRX-mutant SJ-GBM2 cells and immunocytochemistry images (right image) demonstrating their punctate nuclear staining of ATRX only in ATRX-wildtype KNS42 cells. (B) C-circle assay results demonstrating DNA from each sample in the presence (+) or absence (−) of Φ29 polymerase; positive staining demonstrates ALT in U251-ATRX^KO cells only. (C) In vitro data showing proliferation of U251 ATRX^KO cells and its isogenic control, as well as KNS42 (ATRX wildtype) and SJ-GBM2 (ATRX mutant), after exposure to increasing doses of irradiation (IR). (D) In vitro mitotic index assay at 1 hour after 10 Gy IR and 16 hours after 4.5 Gy IR demonstrates that U251 (ATRX^KO) and SJ-GBM2 (ATRX mutant) cells display increased proliferation following treatment with IR. All values normalized to respective 0 Gy value. 0 Gy is normalized to 1.0. (E) Real-time analysis of cell cycle transitions using the Incucyte imaging system and the FastFUCCI reporter plasmid shows the U251 ATRX^KO cells return to active cycling 2X faster after 4 Gy IR. [Mean ± SEM for triplicate experiments are shown. *P≤0.05, **P≤ 0.01, ***P≤0.001, and ****P≤0.0001 using 2-Way ANOVA.] For additional data, see also Figure S1.