Fig. 1.

TERRA foci formation in mouse medulloblastoma. (A) (Top panel) Hematoxylin and Eosin staining of a region of a Ptch1^+/− mouse cerebellum with non-tumor (left panel; indicated by an arrow) and tumor tissue (right panel; no. 6850). The normal morphology of cerebellar folia with the IGL (containing the mature granule neurons) and the Purkinje layer containing the Purkinje neurons is seen in left panel. Scale bar: 200 µm. (Lower panel) Sections of cerebellum containing both normal and tumor tissue showing the results of in situ hybridizations with specific digoxigenin-labeled RNA probes for Gli1 and Math1. Note that Gli1 and Math1 are highly expressed in the tumor part of the cerebellum. Asterisk in left panel denotes the normal expression of Gli1 in the normal Bergmann glial cells that form a layer in this, still organized, non-tumor part of the cerebellum. Scale bars: 100 µm. (B) Confocal images of FISH analyses of TERRA expression in sections of the cerebellum of the same mouse as above containing both normal and tumor tissue. Note the presence of TERRA expression (strong red labeling) in the tumor as compared to the low or undetectable expression in the non-tumor cerebellar tissue. Asterisk denote non-specific TERRA signal in the Purkinje layer. RNase A treatment leads to an elimination of the tumor-specific TERRA signal (middle panels). Images were taken with 40× lens (lower panels) to reveal details of TERRA localization in the nuclei. Scale bar: 20 µm. Other images were taken with 20× lens. (C) Histogram comparing relative TERRA fluorescence signal intensity (FU) in non-tumor (black) vs tumor (red) tissue, analyzed by RNA FISH and ImageProPlus software. (D) TERRA expression measured as total mean fluorescence intensity (FU). Values are means ± standard error from three independent experiments. (E) Quantification of cells with ≧1 TERRA signals of mean intensity >40 FU. More than 800 nuclei from at least three independent experiments were counted for quantification. The P-value was calculated using a two-tailed Student's t-test in all cases. (F) Confocal images of FISH analyses of TERRA expression in tumor (top panel) or non-tumor (middle panel) using an Alexa-Fluor-488-conjugated DNA oligonucleotide probe (TAACCC)₇ (top two panels) or a control mutant probe (TAACAC)₇ as a negative control (lower panel). Sections of the cerebellum of the same mouse (no. 7040) as in supplementary material Fig. S1 containing both non-tumor and tumor tissue were used. Scale bar: 20 µm.