Conditional over-expression of PRMT1 reduces FUS-dC-related aggregates.

<p>(A) Tet-on inducible Myc-PRMT1 wt over-expression HEK293 cells, transiently transfected with plasmid encoding HA-tagged FUS-dC, were simultaneously treated with 1 µg/ml tetracycline (Tet+) or mock (Tet−) for 24 hours. Then samples were fixed, co-stained with anti-HA and anti-Myc antibody and followed by the incubation with Alexa488 and Alexa568 conjugated secondary antibody. Samples were then stained with DAPI. Images were obtained using fluorescence microscopy. Bars, 20 µm. Arrows indicate cells harboring HA-FUS-dC-mediated inclusions. (B) The percentage (%) of cells, harboring FUS-dC-mediated inclusions in the presence (Tet +) or absence (Tet −) of tetracycline for 24 hours, was expressed as mean ± S.D. of 4 separate experiments. Cells with at least one visible inclusion were counted as positive. Four dishes were used per experimental group, with at least 100 cells in four random fields being counted on each trial. <i>^* p</i><0.05 vs Tet- (C) Western blot analysis. Tet-on inducible Myc-PRMT1 wt over-expression HEK293 cells, transiently transfected with plasmid encoding HA-FUS-dC, were treated with 1 µg/ml tetracycline (Tet+) or mock (Tet−) for 24 hours. Then samples were lysed and subjected to Western blot analysis with indicated antibodies. (D) Triton X-100-insoluble fraction assay. Tet-on inducible Myc-PRMT1 wt over-expression HEK293 cells, transiently transfected with plasmid encoding HA- FUS-dC or empty plasmid, were treated with 1 µg/ml tetracycline (Tet+) or mock (Tet−) for 48 hours. Then detergent-soluble (Soluble) and detergent-insoluble (Insoluble) proteins were extracted. Same amounts (30 µg) of soluble and insoluble proteins were loaded in each immunoblot and probed with the indicated antibodies. Immunoreactivity was normalized to the amount of actin. Relative intensities of HA-FUS-dC bands are expressed as mean ±S.D. of 4 separate experiments (right graphs). <i>^*p</i><0.05 vs Tet-.