(A) In a scratch wound assay, 143B cells with shRNA KD of ATRX showed faster wound closure than the nonsilenced (NS) control cells (1-way repeated-measures ANOVA, P < 0.0001 for both shATRX-1 and shATRX-2 compared with NS; n = 8 replicates per cell type; 2 experiments). (B) Representative images of scratch wounds at 3 time points. Scale bars: 200 μm. (C) Western blot showing effective CRISPR-Cas9 KO of ATRX expression in the MG-63 human OS cell line. (D) Similarly, in the MG-63 cell line, KO cells showed significantly faster wound closure than WT cells (1-way repeated-measures ANOVA, P < 0.0001; WT, n = 13 replicates; KO, n = 10 replicates; 2 experiments). (E and F) 143B-KO cells showed increased migration and invasion, respectively, in uncoated and Matrigel-coated transwell plates (1-tailed Student’s t test [E] and 2-tailed Student’s t test [F]; uncoated: P = 0.04, n = 8 replicates; Matrigel: P = 0.002, n = 8 replicates; 2 experiments). Scale bars: 100 μm. (G and H) MG-63–KO cells also showed increased migration and invasion, respectively, in uncoated and Matrigel-coated transwell assays (2-tailed Student’s t test; uncoated: P < 0.0001, n = 12 replicates; Matrigel: P < 0.0001, n = 12 replicates; 2 experiments). (I) After 24 hours of growth on a bed of Matrigel, WT cells remain in tight clusters, whereas KO cells form a network of connecting “tubes” or branching networks through the matrix. Scale bars: 100 μm. (J) After 96 hours of growth, the distinct differences in morphology between the 143B WT and KO cells are even more apparent. Scale bars: 200 μm. (K) Luciferase-labeled 143B WT or KO cells were injected into the subperiosteal space of the tibia of SCID-beige mice. ATRX KO correlates with an increased rate of lung metastasis at 1 week after amputation (1-tailed Student’s t test, P = 0.026; WT: n = 5 males and 10 females; KO: n = 6 males and 11 females). (L) Experimental design for orthotopic injections with LUC-labeled cells to study lung metastasis.