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. 2021 Jun 14;49(12):7011–7034. doi: 10.1093/nar/gkab461

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© The Author(s) 2021. 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 License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 9. — (A) Distribution of TAPSLIVR (ADAT amino acids; green curve) and of codons decoded by I34-tRNAs (ADAT codons; orange curve) along the coding sequence of DAG1. Dotted line indicates the threshold of TAPSLIVR enrichment considered significant as defined in (23). The low-complexity TAPSLIVR-rich region containing the Mucin-like domain, and the α and β dystroglycan (DG) subunits are shown. (B) α-DG and β-DG protein levels in HT-29 M6 shCV and shADAT2 cells. Tubulin is used as gel loading control. Quantification of DG bands relative to Tubulin and normalized to shCV cells for each biological triplicate is shown. Note that for each replicate α-DG and β-DG were measured from the same protein extract. ADAT2 levels in each protein extract is shown in Supplementary Figure S6A. (C) DAG1 transcript levels relative to GAPDH in the indicated cell lines. Shown are biological triplicates, their mean and SD. n.s.: not statistically significant (t-test). (D) ADAT2 protein levels in NCI-H292 shCV and shADAT2 cells treated (+) or not (−) with amphiregulin (AREG). Actin is used as gel loading control. Quantification of ADAT2 bands relative to Actin and normalized to shCV cells (−) AREG is shown. See also Supplementary Figure S6B-C. (E) Distribution of TAPSLIVR (ADAT amino acids; green curve) and of codons decoded by I34-tRNAs (ADAT codons; orange curve) along the coding sequence of MUC5AC. Dotted line indicates the threshold of TAPSLIVR enrichment considered significant as defined in (23). (F) MUC5AC transcript levels relative to GAPDH in the indicated cell lines treated (+) or not (−) with AREG. Shown are biological triplicates, their mean and SD. n.s.: not statistically significant (t-test). (G) MUC5AC expression levels upon AREG treatment in NCI-H292 shCV (blue) or shADAT2 (red) cells, quantified by FACS. Only MUC5AC(+) cells are shown. Left panel: representative assay showing number of MUC5AC(+) cells (y-axis) and MUC5AC signal intensity (x-axis). Right panel: MUC5AC mean signal intensity obtained for three biological replicates. The mean of the means and SD is also shown. *: P-value < 0.05 (t-test). See also Supplementary Figure S6D and E. (H) MUC5AC expression in NCI-H292 shCV and shADAT2 cells treated (+) or not (−) with AREG, quantified by confocal microscopy. Left panel: representative confocal microscopy images. MUC5AC (red) and nuclei staining (DAPI, blue) are shown. Scale bar corresponds to 10 μm. Right panel: quantification of microscopy images (n > 30) as those shown in Left panel for the indicated cell lines with (+) or without (−) AREG treatment with their mean and SD. n.s.: not statistically significant. ****: P-value < 0.0001 (t-test).