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. 2019 Oct 4;47(20):10728–10743. doi: 10.1093/nar/gkz798

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© The Author(s) 2019. 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 7. — Rescue of frataxin protein levels in FRDA patient-derived lymphoblasts. (A-D) GM16214 cells (patients’ primary lymphoblasts) were stably transfected with miniSINEUP-FXN variants and empty vector (ctrl). miniSINEUP-FXN (−40/+0 M1-AUG), miniSINEUP-FXN (−40/+4 M76-AUG) and ctrl stable clones were obtained from at least 15 days of G418 selection. (A) Whole cell lysates were analysed by western blotting with anti-FXN and anti-β-actin antibodies. Two representative experiments are shown. First, FXN band intensity was normalized to the relative β-actin. Then, fold change values were calculated normalizing to GM16214 cells. GM16214 cells expressing miniSINEUP-FXN show increased levels of endogenous FXN protein. (B) Average fold change of FXN protein levels. Columns represent mean ± S.E.M. of n ≥ 4 independent experiments; ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (one-way ANOVA followed by Dunnett's post-test). (C, D) The real-time RT-PCR analysis of FXN mRNA and miniSINEUP RNA expression in transfected cells. Columns represent mean ± S.E.M. of n≥4 independent experiments. Variation in both target and miniSINEUP mRNA expression among samples are not statistically significant (one-way ANOVA followed by Dunnett's post-test). (C) FXN transcripts were quantified, using human GAPDH (hGAPDH) expression as the internal control. The FXN/hGAPDH ratio for the GM16214 sample was set as a baseline value to which all transcripts levels were normalized. Unchanged FXN mRNA levels are shown. (D) miniSINEUP transcripts were quantified, using the hGAPDH expression as the internal control. The miniSINEUP/hGAPDH ratio for −40/+0 M1-AUG sample was set as a baseline value to which all transcripts levels were normalized.