Zebrafish brain tumors are ALT. (A) Representative C-Circle assay by dot blot in one control and one brain tumor compared with telomerase positive HeLa cells and ALT U2OS cells. Reactions without phy29 polymerase (–θ29) were included as a control. (B) Quantitation and analysis of 4 C-Circle assay dot blot. Determination of ALT amount was calculated after subtracting global background and specific –θ29 signal. a.u.: arbitrary unit. (C) C-Circle assay quantified by telomere qPCR. Data are represented as amount of C-circles, normalized to telomere content (TC) and single copy gene (rps11). HeLa and U2OS were added as a reference. Ctrl n = 5; RAS n = 7. The dashed line indicates the level above which ALT activity is considered significant. Whiskers box plots represent median: min to max values. (D) Schematic drawing to describe the procedure for 2-color CO-FISH and the interpretation of telomere status based on the signals. (E) Two-color CO-FISH of a representative metaphase nucleus derived from a RAS brain tumor cell (scale bar 5 μm). The right panels (D) show details of telomeres with T-SCE (white arrows), signal free ends, multimeric signal and/or ECTR (yellow arrows) (scale bar 1 μm). (F) Quantitation of telomeric defects revealed in RAS brain (n = 88 chromosomes) compared with Control brain (n = 56 chromosomes). T-SCE: Telomere Sister Chromatid Exchange; ECTR: Extra-Chromosomal Telomeric Repeat; CO-FISH: Chromosome orientation FISH. Multiple t-test, Holm Sidak methods, *p < 0.05. Bars represent mean ± SEM. See also Supplementary Figure 2.
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