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. 2017 Feb 7;7:42056. doi: 10.1038/srep42056

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Copyright © 2017, The Author(s)

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(A) Significant reduction of the mouse TERRA transcriptional levels on chromosomes 18q and 10q were found in Rb1^+/Δ19 osteoblasts (squared) when compared to their wild-type counterparts (filled column). Error bars show the SD of 2 biological replicates with 2 technical replicates each. RNA-FISH of TERRA in murine osteoblast cells reveals different strengths of labeling of TERRA foci between wild-type and Rb1 haploinsufficient cells. The number of foci were counted in 250 cells of each genotype. Upper panels show a representative image of the cell nuclei with TERRA foci present in wild-type osteoblasts (left) and distribution histogram (right). Lower panels show a representative image of the TERRA foci present in Rb1 haploinsufficient osteoblasts (lower left) and the distribution histogram (lower right). Mean (bars) and SDs (error bars) were obtained from two technical replicates. Yellow is the TERRA signal stained with a strand specific telomeric probe labeled with Cy3 and blue is the DAPI staining the cell nucleus. (C) Endogenous TERRA expression appears modulated by Rb1 levels on murine chromosomes 18q and 10q. A reduction of the Rb1 protein with lentiviral infection expressing shRB1 led to a further decrease of TERRA levels in the Rb1^+/Δ19 osteoblasts. A lentiviral construct (LV-RB1) overexpressing Rb1 rescued the haploinsufficient phenotype leading to an increase in endogenous TERRA expression. Error bars show the SD of 2 biological replicates with 2 technical replicates each (D) Endogenous TERRA expression in human U2OS cells was modulated by Rb1 expression levels on chromosomes 15q and 10q. TERRA levels were reduced after transient transfection with a shRB1 vector. In the same manner transient overexpression of LV-RB1 led to a significant increase of TERRA levels in the human osteosarcoma cell line. Error bars show the SD of 3 technical replicates. Significance of all experiments assessed with one-way ANOVA.