Zebrafish brain tumors exhibit elevated TERRA expression. (A) Schematic drawing describing the generation of TERRA from subtelomeric regions. Cen = centromere, RNA pol II = RNA polymerase II. (B) Representative pictures of TERRA RNA-FISH in cell nuclei (blue) from control (ctrl), tumor (RAS) and RNase treated tumor cells (+RNAse). TERRA foci are shown in magenta. Scale bar: 5 μm. (C) Scatter plot of TERRA RNA-FISH expression measured as the number of foci per nucleus in Control (Ctrl, n = 55) and tumor (RAS, n = 51) nuclei. Representative data from one of three experiments are shown (**p = 0.013). Bars represent mean ± SEM. (D) Scatter plot of TERRA RNA-FISH signals measured as fluorescent intensity per spot per nucleus in control (Ctrl) and RAS brains. Representative data from one of three experiments are shown (***p < 0.001). Bars represent mean ± SEM. (E) TERRA expression measured by dot blot from total RNA (500 ng) of a control and a RAS brain tumor (upper panel); to control for RNA loading, hybridization with an actin RNA probe was performed (lower panel). (F) qPCR analysis of TERRA expression in brain tumors and controls. Values were normalized first to rps11 mRNA levels and then related to TERRA expression in control brains (n = 3). TERRA expression levels were also evaluated in HeLa and U2OS cells (n = 2 samples each) for comparison. Reactions without reverse transcriptase were performed as controls for TERRA quantification (-RT, ND: not detectable). Control bars represent mean ± SEM.
|
