Loss of atrx in p53/nf1-depleted zebrafish tumors results in tert downregulation, ALT and disturbed PRC2 function.(A) FPKM values show a significant downregulation of tert in p53/nf1/atrx-deficient fish compared to p53/nf1-kockout control fish; values were compared with the two-tailed unpaired t-test; n = 3, p = 0.0387. (B) Fluorescence in situ hybridization (FISH) using a Cy3-conjugated telomere-specific probe (TelC-Cy3, red) reveals elongated telomeres visible as large irregularly shaped spots in tumors with a knockout of p53, nf1 and atrx compared to atrx-wildtype control tumors, consistent with alternative lengthening of the telomeres; DNA stained with DAPI (blue); scale bars: 10μm; n = 6. (C) Quantification of relative telomere area in FISH images, normalized to DAPI signal area; values were compared with the two-tailed unpaired t-test; *p = 0.0151; n = 6. (D) Quantification of the relative number (#) of all detectable telomere spots in FISH images, normalized to DAPI signal area; values were compared with the two-tailed unpaired t-test; *p = 0.042; n = 6. (E) Quantification of the relative number (#) of telomere spots larger than ~1.5μm2 in FISH images, normalized to DAPI signal area; values were compared with the two-tailed unpaired t-test; *p = 0.0013; n = 6. (F) RNA-seq analysis demonstrates significantly increased expression of PRC2-target gene sets in atrx-deficient tumors; group 1: p53-/-, nf1b-/-, nf1a+/-, atrx+/+ (control); group 2: p53-/-, nf1b-/-, nf1a+/-, atrx+/-; n = 3.
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