FIGURE 2.
TDP-43-induced death is associated with the increase of Bim expression and the decrease of Bcl-xL expression. A and B, NSC34 cells, seeded on 6-well plates at 1 × 105 cells/well, were co-infected with LacZ or TDP-43 virus at a m.o.i. of 400 in association with cre-recombinase or LacZ virus at a m.o.i. of 40. After infection, some cells were co-incubated with 100 μm Z-Asp-CH2-DCB. At 24 h, medium was replaced with DMEM/N2 supplement with or without 100 μm Z-Asp-CH2-DCB. 24 h after medium replacement, LDH release was measured (A). Immunoblot (IB) analyses were performed with antibodies to cleaved-poly(ADP-ribose) polymerase (PARP), Bim, Bcl-xL, and TDP43-C (B). The ratio of immunoreactive density of Bim and Bcl-xL to that of GAPDH was measured using ImageJ software. C and D, NSC34 cells, seeded on 6-well plates at 1 × 105 cells/well, were infected with LacZ or TDP-43 virus at a m.o.i. of 400. These viruses are able to express proteins without co-expression of cre-recombinase. 24 h after infection, medium was replaced with DMEM/N2 supplement. 24 h after medium replacement, immunoblot (IB) analyses were performed with antibodies to TDP43-C, cleaved caspase-3, Bim, Bcl-xL, Bcl-2, and Mcl-1 (C) and quantitative real-time PCR analysis for Bim, Bcl-xL, Bcl-2, and Mcl-1 were performed (D). Data were analyzed with Student's t test. E, NSC34 cells, seeded on 48-well plates at 2.5 × 104 cells/well, were transfected with 0.1 μg/well of the Bim-luc vector in association with 0.1 μg/well of pEF1-Myc/His-vec or pEF1-TDP-43. At 48 h after transfection, luciferase activity was measured by dual-luciferase assays. Data were analyzed with Student's t test. Immunoblot analysis was performed with TDP43-C antibody.