Fig 2: ATRX-deficient high-grade glioma cells are sensitive to multi-targeted RTK and specific PDGFR inhibitors. (A–E) Relative cell viability of U-251 (EV and ATRX KO) high-grade glioma cell lines treated with increasing doses of the indicated drug. The IC50 values were calculated from the dose–response curve determined by GraphPad Prism. Data shown correspond to a representative experiment (with technical triplicates) out of three biological replicates. SEMs from each data point are indicated. Significance was assessed by F-test and the p-value for each dataset is shown. Representative images of EV and ATRX KO cells after 48 h of treatment with DMSO or the indicated drug concentrations are shown.

Fig 3: Loss of ATRX changes the enrichment of a subset of proteins at telomeres.A: Correlation between BioID bait proteins. RAP1 BioID experiments strongly correlated with one another independently of ATRX expression. B: Relative ATRX detection by RAP1 BioID in ATRX-expressing and isogenic cells where ATRX expression was knocked-down (KD) using shRNA or knocked-out (KO) using CRISPR-Cas9. C: Fold change of prey proteins identified by RAP1 BioID (BFDR = 5%, SAINT [103]) in ATRX-expressing vs. ATRX-null HEK293 Flp-In cells. Labeled proteins had =2.5 average spectra and =1.5-fold change between conditions. Proteins labeled in the scatter plot remained significant when NLS-BirA* was used as a control. Inset box represents additional proteins identified with NLS-BirA* as a bait (less stringent).

Fig 4: ATRX associates with chromatin under conditions that induce CFS expression. (A) IF analysis of ATRX (green) in G2 cells. HeLa cells were treated with DMSO or APH (0.2 µM) for 20 h, and G2 cells were selected based on DAPI profile. Representative images are shown. (B) Analysis of the number of ATRX and FANCD2 foci across the cell cycle. HeLa cells were incubated with DMSO or APH (0.2 µM) for 20 h and EdU (20 µM) was incorporated for the last 30 min. Analysis was performed using quantitative image-based cytometry (QIBC). DAPI and EdU profiles were used to determine cell cycle stages (G1, early, mid and late S and G2). Z-stack images were acquired to determine the number of foci per cell. The average number of ATRX foci per cell in each stage is illustrated in the box plots for vehicle (blue) or after APH treatment (green). Data shown corresponds to the average of >700 cells per condition. Means, SDs, maximum and minimum values are indicated. Significance was evaluated with unpaired t-test ****P = 0.0001. (C) IF analysis for colocalization of ATRX (green) and FANCD2 (red) in G2 cells. HeLa cells were treated with DMSO or APH (0.2 µM) for 20 h. Two different colocalization patterns are indicated: complete foci overlap (middle row and indicated by white arrows) and adjacent foci (bottom row). Representative images are shown. Selected regions are magnified. (D) Quantification of FANCD2 and ATRX colocalizing foci per G2 cell. Data shown correspond to biological duplicates of 300 cells per condition. Means and SDs are indicated. Significance of the total number of colocalizing foci was evaluated with unpaired t-test ****P = 0.0001. (E) Quantification of the percentage of FRA16D loci colocalizing with ATRX foci. HeLa cells were treated with DMSO or APH (0.2 µM) for 20 h and arrested at G2. Data shown corresponds to biological triplicates of 300 cells per condition. Means and SDs are indicated. Significance was evaluated with unpaired t-test. *P = 0.05. (F) Example of ATRX IF (red) colocalizing with FRA16D (green) in G2 cells. Selected regions are magnified.

Fig 5: ATRX proteome overview and proposed model.A: Protein associations with ATRX, FAM207A, and CCDC71. Pale grey circles denote previously reported associations, while dark red circles represent newly identified proximal associations. Blue circles represent previously reported associations also identified by our BioID. Image was built using ProHits Viz [104] and the BioGrid database [52], and manually organized. B: Model proposing new biological roles for ATRX (other than histone deposition), mediated through proximal associations with FAM207A, CCDC71, and SLF2.