1. A Drosophila cell culture model to study VAP(P58S) aggregation.
A Drosophila cell culture model to study VAP(P58S) aggregation. (A) VAP:GFP and VAP(P58S):GFP, when expressed in S2R+ cells, allow efficient visualization of VAP protein in the cell by epifluorescence. (B,C) In stable cell lines, expression of VAP(P58S):GFP, under an inducible metallothionein promoter results in aggregation (C), unlike wild-type VAP:GFP (B). GFP is visualized by epifluorescence and chromatin by DAPI, post-fixation. Arrows indicate cells expressing VAP:GFP (B) or VAP(P58S):GFP (C). (D) A super-resolution image, using Ground State Depletion microscopy, showing GFP inclusions formed in cells expressing VAP(P58S):GFP but not in VAP:GFP. (E) VAP(P58S):GFP protein levels in cells increase with increasing CuSO4 concentration at 24 h post-induction. (F) The increase in the fraction of S2R+ cells showing GFP-positive inclusions increases with increasing CuSO4 concentration. At 500 mM CuSO4, inclusions significantly increase between 24 h and 36 h. Student's t-test (*P<0.05). (G) A linear correlation between the fraction of cells showing aggregation, measured using microscopy, plotted against relative VAP(P58S):GFP protein levels, as quantified by western blotting, at 24 h post-induction. Error bars indicate s.d.
Kriti Chaplot et al. Dis. Model. Mech. 2019;12:dmm033803
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