Figure 1: Exogenously expressed ATRX represses the ALT pathway. | Nature Communications

Figure 1: Exogenously expressed ATRX represses the ALT pathway.

From: Suppression of the alternative lengthening of telomere pathway by the chromatin remodelling factor ATRX

Figure 1

(a) Immunoblot showing expression of ATRX in U-2 OSATRX cell line on addition of 0.4 μg ml−1 doxycycline for the specified number of days. Alpha tubulin is shown as a loading control. (b) Chromatin Immunoprecipitation showing that on addition of 0.4 μg ml−1 doxycycline for 4 days the exogenously expressed ATRX is targeted to telomeres. Immunoprecipitated DNA was slot blotted and probed with a 32P-labelled TTAGGG probe. (c) Induction of ATRX by doxycycline leads to a fall in C-circles which is reversed on withdrawal of doxycycline at day 5 (shown as a dashed line). (d) Representative immunofluorescence images showing the presence of ALT-associated PML bodies (APBs) in untreated U-2 OSATRX cells and the reduction in APBs on induction of ATRX expression by addition of 0.4 μg ml−1 doxycycline for 4 days. A 10-μm scale marker is shown. APBs (e) and TIFs (f) are scored as a direct co-localization between either TRF2 and PML for APBs, or between TRF2 and 53BP1 for TIFs, with the chart showing the average number of APBs or TIFs in U-2 OSATRX cells with and without ATRX expression. Co-localizing foci were scored using the JACoP plugin for ImageJ. Number of cells scored (n) is shown in parentheses. Statistical significance was determined using a Mann–Whitney test. (g) Terminal restriction fragment length analysis using a TTAGGG probe showing progressive telomere shortening on expression of ATRX. (h) Representative image of chromosome orientation fluoresence in situ hybridization (CO-FISH). G-rich telomeric DNA is stained in red. A crossover event is depicted with a white arrow, a 10-μm scale marker is shown. (i) Quantitation of CO-FISH in U-2 OSATRXcells before and after ATRX induction for 4 days. Telomere sister chromatid exchange (TSCE) was scored as a percentage of exchanges per metaphase spread. Error bars denote s.e.m. Over 2,000 telomere ends were scored for cross-overs per treatment group. Statistical significance was determined using a Mann–Whitney test.

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