Extended Data Fig. 1: Confirmation of telomeric 8oxoG induction in FAP-TRF1 expressing cells.
From: Telomeric 8-oxo-guanine drives rapid premature senescence in the absence of telomere shortening
(a) Representative images of FAP-mCER-TRF1 colocalization with telomeres in BJ (top panel) and RPE (bottom) clones expressing FAP-TRF1 visualized by anti-mCER staining (red) and with telo-FISH (green). Scale bar = 10 μm. (b) Immunoblot for TRF1 in whole cell extracts from hTERT BJ and RPE cells with and without FAP-mCER-TRF1 expression. TRF1 antibody detects both exogenous and endogenous TRF1 while mCER antibody detects exogenous only. (c) Quantification of YFP-XRCC1 signal intensity at telomeric foci as shown in (Fig. 1a) normalized to CFP signal. Box plots represent median and 25th to 75th percentiles, and whiskers represent the 1st to 99th percentiles. Data derived from the indicated n number of foci analyzed. Statistical analysis was by one-way ANOVA (ns = not significant, * p < 0.05, ***p < 0.001). (d) Quantification of number (#) of FAP-mCer-TRF1 foci per cell by direct mCer visualization of untreated cells (UT) or 10 min after dye + light (DL 10’). Error bars represent the mean ± s.d. from the indicated n number of nuclei analyzed. Statistical analysis by two-tailed t-test (p value was not significant). (e) Quantification of percent YFP-XRCC1 positive telomeres per nuclei after 10 min dye + light with pretreatment of 100 µM NaN3 for 15 min prior to light exposure (NaAz) or with no pretreatment (-). Error bars represent the mean ± s.d. from the indicated n number of nuclei analyzed. Statistical analysis by two-tailed t-test (**p < 0.01). (f,g) Detection of 8oxoG in telomeres. Genomic DNA isolated from RPE (f) and BJ (g) FAP-TRF1 cells following no treatment, 5 or 20 min dye + light or 40 mM KBrO3, was treated with FPG glycosylase, and then treated with S1 nuclease (+) or not (-) as indicated. Intact and cleaved telomere restriction fragments were separated by PFGE, and telomeres were detected by Southern blotting. NE = no enzyme treatment for reference from UT cells. (h) The percent of cleaved telomeres was calculated and normalized to UT samples to quantify a fold change in telomere cleavage. For RPE the difference between -S1 and +S1 was used, and normalized to UT cells.
