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. 2019 Aug 29;9:12516. doi: 10.1038/s41598-019-49032-0

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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7SK snRNA and LARP7 mRNA and protein amounts are decreased, while mRNA and protein levels of HEXIM1 are increased in patient cells. (a) Quantification of the 7SK snRNA by RT-qPCR. GAPDH mRNA was used as an internal control, and the amount of each analysed RNA relative to GAPDH mRNA is presented. The mean of four (patient and two healthy individuals) or three (LARP7 KO) independent experiments ± SD is given. *p ≤ 0.05; ***p ≤ 0.001; ****p ≤ 0.0001 by one-way ANOVA followed by Bonferroni post-hoc test for multiple comparison. (b) Immunoblot of lysates obtained from patient, control 1 and patient’s mother fibroblast cultures from three different passages. Endogenous LARP7 and HEXIM1 were monitored with specific antibodies, and anti-Tubulin antibody was used to control for equal loading. Representative blots are shown. Full-length blots are presented in Figs S2 and S3. (c) Band intensities of LARP7 protein were quantified using a chemiluminescence imager, and LARP7 protein was normalized to Tubulin. The mean of six independent experiments ± SD is given. ****p ≤ 0.0001 by one-way ANOVA followed by Bonferroni post-hoc test for multiple comparison. (d) Quantification of LARP7 transcript amount by RT-qPCR. GAPDH mRNA was used as an internal control, and the amount of each analysed RNA relative to GAPDH mRNA is presented. The mean of four (patient and two healthy individuals) or three (LARP7 KO) independent experiments ± SD is given. **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001 by one-way ANOVA followed by Bonferroni post-hoc test for multiple comparison. ns, not significant. (e) Band intensities of HEXIM1 protein were quantified using a chemiluminescence imager, and HEXIM1 protein was normalized to Tubulin. The mean of six independent experiments ± SD is given. ***p ≤ 0.001 by one-way ANOVA followed by Bonferroni post-hoc test for multiple comparison. (f) Quantification of HEXIM1 transcript amount by RT-qPCR. GAPDH mRNA was used as an internal control, and the amount of each analysed RNA relative to GAPDH mRNA is presented. The mean of four (patient and two healthy individuals) or three (LARP7 KO) independent experiments ± SD is given. **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001 by one-way ANOVA followed by Bonferroni post-hoc test for multiple comparison. (g) Quantification of HEXIM1 transcript amount by RT-qPCR after treatment of patient and LARP7 KO fibroblasts with 10 nM flavopiridol for 30 min. GAPDH mRNA was used as an internal control, and the amount of each analysed RNA relative to GAPDH mRNA is presented. The mean of three independent experiments ± SD is given. **p ≤ 0.01 by one-way ANOVA followed by Bonferroni post-hoc test for multiple comparison.