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. 2014 May 1;10(5):e1004318. doi: 10.1371/journal.pgen.1004318

Figure 3. Over-expressed RNase H1 resolves R-loops formed on FXN expanded repeats in HEK293 cells.

Figure 3

A. Diagram of the FXN-Luc gene, containing 6 (FXN-Luc) or 310 GAA repeats (FXN-GAA-Luc), integrated on the chromosome 1 of HEK293 cells. Frataxin gene was fused to the luciferase at the beginning of the FXN exon 5. Black boxes are exons, white boxes are 5′ and 3′UTRs, lines are introns, red triangle is (GAA)n expansion. TSS is the transcriptional start site. qPCR amplicons are shown below the diagram. Numbers indicate the distances to TSS in kilobases. B. Size of GAA expansion determined by PCR analysis on genomic DNA from FXN-Luc and FXN-GAA-Luc cell lines, using GAA104F and GAA629R primers. PCR products were run on 1% agarose gel. M denotes the marker lane. FXN-Luc and FXN-GAA-Luc cells contain endogenous wild type FXN gene [23], giving rise to the PCR product of 0.5 kb. C. FXN and γ-actin nascent RNA levels in FXN-Luc (white bars) and FXN-GAA-Luc (black bars) HEK293 cells, determined by RT-qPCR and normalised to 5S rRNA. The level of FXN and γ-actin nascent RNA in FXN-Luc cells was taken as 1. LucR primer was used for the reverse transcription reaction. qPCR was carried using in4F and ex5R primers, shown in A. D. DIP analysis on FXN-Luc gene in FXN-Luc (white bars) and FXN-GAA-Luc (black bars) HEK293 cells using RNA/DNA hybrid-specific S9.6 antibody. E. RT-qPCR analysis of RNase H1 mRNA from FXN-Luc and FXN-GAA-Luc cells, treated with control and RNase H1 siRNAs. Values are normalised to GAPDH mRNA and are relative to FXN-Luc cells, treated with control siRNA. F. DIP analysis on FXN-Luc gene in FXN-Luc and FXN-GAA-Luc HEK293 cells, treated with control and RNase H1 siRNAs. G. Western blot analysis of 50 µg of protein extracts obtained from FXN-Luc and FXN-GAA-Luc cells transfected with Flag and RNase H1-Flag expression plasmids. Western blot was probed with anti-RNase H1 antibody. * denotes endogenous RNase H1 protein. H. DIP analysis on FXN-Luc gene in FXN-Luc and FXN-GAA-Luc HEK293 cells transfected with Flag or RNase H1-Flag expression plasmids. Bars in C–F and H represent the average values from at least three independent experiments +/− SEM.

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