Fig. 4. DAPK1 exacerbates rotenone-induced toxic synucleinopathy in SH-SY5Y and MEF cells. (A) SH-SY5Y cells were either mock-transfected or transfected for 24 h with a plasmid encoding FLAG-DAPK1-WT, and treated with 100 nM rotenone for additional 24 h. Cell lysates were fractionated into 1% Triton X-100-soluble and -insoluble fractions, followed by immunoblotting with the indicated antibodies. (B) DAPK1-WT or DAPK1-KO MEFs were treated with either DMSO (control) or 100 nM rotenone for 24 h. Cells were lysed with detergent-free lysis buffer and the protein samples were analyzed by native PAGE. (C) After DAPK1-WT or DAPK1-KO MEFs were mock transfected or transfected for 24 h with a plasmid encoding FLAG-DAPK1-WT, cells were treated with either DMSO (control) or 100 nM rotenone for an additional 24 h. Representative confocal images of immunostaining of endogenous α-synuclein (green) and FLAG-DAPK1 (red) are shown in the upper panel. In the lower panel, the number of cells showing intracellular and visible α-synuclein aggregates were counted among ~100 cells, and this was repeated five times. Data represent the mean±SEM of five independent experiments (**p≤0.01). (D) Where indicated, DAPK1-WT or DAPK1-KO MEFs were mock-transfected or transfected for 24 h with a plasmid encoding FLAG-DAPK1-WT, and treated with either DMSO (control) or 100 nM rotenone for additional 24 h. Representative confocal images of immunostaining of pS129-α-synuclein (green) and FLAG-DAPK1 (red) are shown in the upper panel. In the lower panel, the number of cells showing intracellular and visible α-synuclein aggregates was counted in approximately 100 cells, and the counting was repeated five times. The data represent the mean±SEM of five independent experiments (**p≤0.01). (E) Where indicated, SH-SY5Y cells were transfected for 24 h with plasmids encoding FLAG-DAPK1-WT or FLAG-DAPK1-K42A, or α-synuclein (SNCA) siRNA alone or in combination. Cells were then treated with either DMSO (control) or 100 nM rotenone for an additional 24 h. Cell viability was measured using Cell Counting Kit-8. Data represent the mean±SEM of five independent experiments (**p≤0.01).
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