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. 2018 Apr 18;46(11):5504–5524. doi: 10.1093/nar/gky263

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© The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 4. — TNBL is a long, non-polyA, nuclear and stable transcript. (A) TNBL RT qPCR of oligo(dT) or random primed cDNA from HCT116 (left) and Caco-2 (right) AZA and TSA treated cells. Control RNAs: PUM1 as a polyA containing RNA and 18S rRNA as a non polyA containing RNA. (B) Relative enrichment (in %) of TNBL, XIST and PUM1 transcripts in nuclear insoluble, nuclear soluble and cytoplasmic RNA fractions from AZA and TSA treated Caco-2 cells analyzed by RT-qPCR. (C) TNBL stability relative to GAPDH RNA measured by RT qPCR at several time points in LS174T cells treated with Actinomycin D (ActD) during a 6 h time course using two NBL2 specific primer sets (1 and 3). PUM1 and MALAT1 were used as controls of mature RNA, and 45s rRNA of pre-mature RNA. (D) Left: TNBL sense probe 1 Northern blot on RNA from untreated (NT), AZA and AZA/TSA treated Caco-2, HCT116 and TOV112D cells. RNA from mouse C2C12 myoblasts treated with AZA/TSA was used as a negative control. Middle: TNBL sense probe 2 Northern blot on RNA from HCT116 untreated (NT) and AZA/TSA treated, DKO untreated and TSA treated. Negative control: chromosome 9 human-rodent somatic cell hybrid treated with AZA/TSA. Right: TNBL sense probe 2 Northern blot on RNA from LS174T cells. Sample integrity and equal loading was assessed with 18s and 28s rRNA methylene blue staining. (E) NBL2 expression analyzed with RT qPCR in human rodent somatic cell hybrids untreated (NT), AZA treated, and AZA/TSA treated. (F) Northern blot on RNA from chromosome 13, 14, 15 and 21 human rodent somatic cell hybrids untreated and AZA/TSA treated. Sample integrity and equal loading was assessed with 28s rRNA methylene blue staining. (G) Scheme of DNA fragments cloned into pGL3-basic vector driving luciferase gene expression (LUC). Promoter activity was analyzed in DKO cells. NBL2 array is represented 3′ to 5′. 16 different constructs were analyzed: 10 different NBL2 monomer sequences (1.4 kb each); a monomer 21 (928 bp, truncated monomer); a monomer 21 with adjacent transposon elements (TE1: L1MD2-MLT1D); and four fragments encompassing distinct transposon element upstream regions: TE1 (L1MD2-MLT1D), TE2 (MLT1D-MER70-int), TE3 (LTR7-MER70A), TE4 (LTR7). The graph shows relative luciferase activity of tested fragments compared to empty vector. P = 3.9E–03, as evaluated by Mann-Whitney-Wilcoxon test.