Fig. 6: miRNA strategy to silence Lsd1 and Prmt6 in vivo. | Nature Communications

Fig. 6: miRNA strategy to silence Lsd1 and Prmt6 in vivo.

From: LSD1/PRMT6-targeting gene therapy to attenuate androgen receptor toxic gain-of-function ameliorates spinobulbar muscular atrophy phenotypes in flies and mice

Fig. 6: miRNA strategy to silence Lsd1 and Prmt6 in vivo.The alt text for this image may have been generated using AI.

a Western blots of LSD1 and PRMT6 in MN1 cells transfected with vectors expressing scramble amiR or amiR to silence Lsd1 and Prmt6. Shown is one experiment representative of three biological replicates. b Cell viability assay in MN1 cells expressing either AR24Q or AR100Q transfected as indicated (amiR-Lsd1#1, amiR-Prmt6#6) and treated with DHT (10 nM, 48 h; n = 3 biological replicates). c Schematic of AAV9 vector expressing GFP and amiR-Lsd1#1/Prmt6#6 (amiR-Lsd1/Prmt6). ITR, inverted terminal repeat; WPRE, woodchuck hepatitis virus post-transcriptional regulatory element; pA, poly-adenylation site. Figure was in part generated using pictures from Servier Medical Art licensed under a Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0/) and in part created with Biorender.com. d Biodistribution of viral particles in different tissues of 4-week-old WT mice (Scr-GFP-2E10 virions: n = 3 male mice; Scr-GFP-8E10 virions: n = 3 male mice for liver, heart and quadriceps; n = 2 male mice for spinal cord. e Western blots of GFP expression in the indicated tissues of WT mice transduced by AAV9-amiR. Shown is one experiment representative of three biological replicates in three mice. f RT-PCR analysis of transcript levels of Lsd1, Prmt6, mouse Ar (mAr), and human AR (hAR) in the quadriceps muscle of 13-week-old AR100Q mice with or without amiR-Lsd1/Prmt6 (Lsd1 n = 8 AR100Q and n = 7 AR100Q amiR-Lsd1/Prmt6; Prmt6 expression: n = 8 AR100Q mice and n = 8 AR100Q amiR-Lsd1/Prmt6; mAr and hAR: n = 4 mice/treatment). g Western blots of LSD1 and PRMT6 in the skeletal muscle of 13-week-old AR100Q mice treated with or without amiR-Lsd1/Prmt6. Quantification is shown at the bottom. *LSD1 p = 0.03, *PRMT6 p = 0.023 (LSD1 expression: n = 6 mice/treatment; PRMT6 expression: n = 3 mice/treatment). Shown is one representative experiment. GFP, LSD1, and PRMT6 were detected with specific antibodies, and β-Tub and CNX were used as loading controls. Graphs show mean ± SEM; two-tailed Student t-test. Source data are provided as a Source data file.

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