FIGURE 4.

Impairment of the autophagy in TDP-43-depleted cells. A, Western blotting of cell lysates prepared from Neuro2A cells transfected with scRNA oligonucleotide (lanes 1, 3, 5, and 7) or TDP-43 siRNA-1 oligonucleotide (lanes 2, 4, 6, and 8). The cells were grown in either complete medium or serum-free medium. Data from duplicated experiments are shown. Impairment of the autophagy in TDP-43-depleted cells was evidenced by the switch of the relative abundance of LC3-II/LC3-I in lanes 2, 4, 6, and 8 relative to lanes 1, 3, 5, and 7, respectively. B, Neuro2A cells were transfected with scRNA oligonucleotide (lanes 1, 3, 5, and 7) or Atg7 siRNA oligonucleotide (lanes 2, 4, 6, and 8). Data from duplicated experiments are shown. Note the similar patterns of the relative abundance of LC3-II/LC3-I in A and B. The bottom panels in A and B show the quantitative analysis of the LC3-II/LC-I ratios. *, p value < 0.05; **, p value < 0.01. C, electron microscopic analysis of Neuro2A cells transfected with the scRNA oligonucleotide (top panels) or siRNA-1 oligonucleotide (bottom panels) for 48 h and serum-starved post-transfection. The marked areas in the left two panels were magnified to show the autophagosomes (arrows). Cells treated with siRNA-1 oligonucleotide exhibited significantly less number of autophagosomes when compared with cells treated with the scRNA oligonucleotide. The histogram shows the comparison of the mean numbers of autophagic vacuoles (AVs) per EM field in the two sets of samples (n = 3 for each). ***, p value < 0.001.