For testing C9ORF72 iPSN sensitivity to glutamate-induced excitotoxicity, healthy control and C9ORF72 iPSNs were treated with various concentrations of L-glutamate (1, 3, 10, 30, 100 μM) for 2–8 hr. At the appropriate time point, cells were incubated with 1 μM
propidium iodide and 1 μM calcein AM (Invitrogen) for 30 min to visualize dead and live cells, respectively. For ASO rescue experiments, iPSNs were treated with ASOs for 72 hr and then treated with L-glutamate prior to dead/live cell quantification. ADARB2 fusion protein was expressed and purified from Rosetta II cells following gateway expression system (Invitrogen) and GSTrap HP column purification. Increasing concentrations of purified protein was incubated Birinapant in vitro MK-2206 purchase with 10 nM Cy5-labeled RNA. The fraction of RNA shifted, due to ADARB2 binding, was densitometrically quantified in ImageJ (NIH). Z-stack images were
taken on a Zeiss Axioimager with the Apotome tool or a Zeiss LSM510-meta single-point laser scanning confocal microscope matched exposure times or laser settings and normalized within their respective experiment. Statistical analysis was performed using the Student’s t test or one-way analysis of variance with the Turkey’s or Dunnet’s post-hoc test and the Prism 6 software (GraphPad Software, Inc.). Additional details are provided in the Supplemental Experimental Procedures. C.J.D designed, performed, and analyzed the experiments and the OME ASO; maintained, treated, and characterized the C9ORF72 fibroblasts and iPSNs; and wrote the manuscript.
P.Z. optimized and performed Southern blot methodologies and characterized repeat lengths of the cell and tissue used in these data sets. J.T.P. aided in iPSN differentiation, iPSN characterization, and siRNA and RNA-FISH experiments. A.R.H. and J.W. generated and purified ADARB2 protein and performed the EMSA experiments. N.A.M. analyzed and categorized microarray data. S.V. maintained C9ORF72 cell lines, aided in RNA isolation, tested antibody specificity, and optimized and performed the RNA-coIP experiments. E.L.D. differentiated and maintained all iPSCs cells to the neuronal stage and quantified iPSC differentiation efficacy. E.M.P. performed the proteome array assay. B.H. performed imaging analysis for proteome array hits. D.M.F. aided in blinded imaging and gene expression Florfenicol analysis. N.M. and P.T. provided fibroblast or iPSC lines. B.T. screened human tissue for C9ORF72 expanded repeat and provided critical input for experimental design and strategies. F.R. and C.F.B. designed and provided MOE ASOs and performed initial ASO screening as well as providing input on experimental ASO strategies. S.B. provided the proteome array and aided in analysis and interpretation. R.S. and J.D.R. oversaw project development, experimental design, data interpretation, and manuscript writing. This work was funded by grants from NIH (J.D.R.), P2ALS (J.D.R.), Muscular Dystrophy Association (J.D.R.