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sCAGs induce post-transcriptional gene silencing in genes with CTG regions.

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posted on 2012-02-23, 01:56 authored by Mónica Bañez-Coronel, Silvia Porta, Birgit Kagerbauer, Elisabet Mateu-Huertas, Lorena Pantano, Isidre Ferrer, Manuel Guzmán, Xavier Estivill, Eulàlia Martí

A. Hela cells were cotransfected with firefly luciferase expressing vectors containing the indicated nucleotide sequences in its 3′-UTR, the specific HTT-e1 expressing vectors or the (CAG)7 siRNA and Renilla luciferase plasmid to normalize data. Assays were performed 24 hours after transfection. Data were first normalized to the 100% of luminescence obtained with the control luciferase vector, lacking 3′UTR inserts (n = 3; *p<0.05, p**p<0,01). B,C. Levels of ADORA2A and MEIS2 transcripts in SH-SY5Y cells transfected with normal and expanded HTT vectors. MRIP was used as endogenous control. qRT-PCR was performed in cells fixed 24 hours after transfection. (n = 3; *p<0.05). Values represent the mean fold change with respect to the control, non-transfected cells ± SEM. D. Western blot showing reduced MEIS2 protein levels in differentiated SH-SY5Y expressing expanded HTT RNA 24 hours after transfection. The graph shows the densitometry determination of MEIS2 levels vs β-Actin. Results represent the mean arbitrary optical density change normalized to the mean value obtained in non-transfected cells (n = 4; *p<0.05).

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