1. Characterization of DPR proteins in primary rat neurons and HEK293 cells.
Characterization of DPR proteins in primary rat neurons and HEK293 cells. GFP, GFP-(GR)149, or (PR)175-GFP were expressed in primary rat neurons and HEK293 cells. (A) Images show GFP fluorescence of a single confocal plane in cells stained with DAPI to visualize nuclei and anti-fibrillarin (red) to visualize nucleoli. Scale bar denotes 20 μm. (B) Quantitative analysis of poly-GR/PR localization to fibrillarin-positive nucleoli. Average co-localization from n = 17 for GFP-(GR)149, n = 13 for (PR)175-GFP images in neurons and n = 6 for GFP-(GR)149, n = 6 for (PR)175-GFP images in HEK cells from two independent experiments, mean ± SEM is shown. (C) LDH release assay detects significant toxic effect on (PR)175-GFP but not GFP-(GR)149 expression compared with GFP control in primary rat neurons (DIV7 + 14) (18 replicates from three independent experiments; box plot is shown with 25th percentile, median, and 75th percentile; and whiskers representing minimum and maximum, exact P-values: GFP versus GFP-GR149, P = and GFP versus PR175-GFP; P = in unpaired one-way ANOVA with Dunnett’s posttest). (D) Cell viability of HEK293 cells transfected with GFP, GFP-(GR)149, or (PR)175-GFP measured by XTT assay 3 days after transfection. GFP-(GR)149 and (PR)175-GFP showed significantly reduced viability compared with GFP control (12 replicates from two independent experiments; box plot is shown, exact P-values: GFP versus GFP-GR149, P = and GFP versus PR175-GFP, P = in unpaired one-way ANOVA with Dunnett’s posttest).
Hannelore Hartmann et al. LSA 2018;1:e
© 2018 Hartmann et al.