| Autor: |
Vanderplanck, Céline, Tassin, Alexandra, Ansseau, Eugénie, Charron, Sébastien, Wauters, Armelle, Lancelot, Céline, Vancutsem, Kelly, Laoudj-Chenivesse, Dalila, Belayew, Alexandra, Coppée, Frédérique |
| Rok vydání: |
2018 |
| DOI: |
10.6084/m9.figshare.5786217 |
| Popis: |
DUX4c siRNA design and efficiency evaluation. A. Schematic representation of the DUX4c 3′UTR (positions from Genbank accession number AY500824). The STOP codon, two purine-rich regions which could be used as an alternative 3′end processing (as shown for some histone genes, [97]), the restriction sites EcoRI and AflIII (used for p3kb-DUX4c and 7.5-kb-DUX4c plasmid constructs, [26]) and the localization of the three designed siRNAs are indicated. Full-length DUX4c transcripts were already described in healthy and FSHD muscle cells [27]. B. Different DUX4c RNA ends found following transfection of C2C12 cells with p7.5-kb-DUX4c or in primary FSHD myoblasts (indicated by the asterisk). C. Evaluation of DUX4c knock-down using the three siRNAs shown in A. Human muscle TE671 cells were transfected or not (NT) with pCIneo-DUX4c expression vector (DUX4c) and with or without an siRNA targeting DUX4c as indicated (si1, si2, si3). Protein extracts were prepared 3 days later and analyzed by Western blot with the rabbit anti-DUX4c serum. Actin was used as a loading control. The panel with siRNA1 was previously shown in Ansseau et al. 2009 as part of Additional file 3: Figure S3 to confirm anti-DUX4c serum specificity. NT: not transfected. D, E. DUX4c expression in DUX4c-inducible stable TE671 cells [27] transfected with 20 nM DUX4c siRNA1. Four hours later, DUX4c expression was either induced or not with 1 μg doxycycline. C. Three or 5 days later, proteins were extracted, and 20 μg were separated on a 10% PAGE-SDS gel and transferred to a Western blot. DUX4c and actin were immunodetected. D. DUX4c detection by immunofluorescence (red) on parallel cultures. (PDF 90 kb) |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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