Figure 1.
Alterations in cell morphology and nucleus size caused by treatment with rotenone (0.25 or 2 µM) (x200 and selective enlargement).
Cellular morphological changes and axons alterations were visualized using a light microscope after exposure to rotenone (0.25 or 2 µM). The toxic effect of chronic exposure to rotenone at low-dose levels was maintained for 1.5, 3 and 7 days. With exposure to rotenone (2 µM), cells were cultured for 24 and 36 h. Under the same conditions, cells were stained with Hoechst dye and the nucleus size was observed using a fluorescence microscope. The selective enlargement graph was at the bottom.
Figure 2.
Modulation of cell cycle distribution after exposure to rotenone.
Cell cycle analysis was performed using flow cytometer after propidium iodide (PI) staining of target cell DNA. The proportion of cells in each phase was statistically analyzed. The data were collected from three independent experiments and were expressed as the mean ± SD. (A) The cell cycle distribution was altered in response to different concentrations of rotenone for 36 h. * P<0.05 compared to the control group; ** P<0.05 compared to the 1 µM rotenone group. (B) and (C) Cells were cultured with 0.25 µM and 2 µM rotenone for different time periods. * P<0.05 compared to the control group; # P>0.05 compared to the control group; ** P<0.05 compared to the 24 h rotenone group; “>4N” represents cell DNA contents>4N.
Figure 3.
The cell DNA replication was examined by BrdU incorporation.
The binding fluorescence intensity of FITC by flow cytometer determined the initiation of DNA replication. R2 represents cells are under replication. Small and big arrow represents that cells continues DNA replication with DNA contents of 4N.
Figure 4.
DNA poly β, cyclin E and cyclin D expression were detected after treatment with low and high concentrations of rotenone.
The expression of DNA poly β, cyclin E and cyclin D were investigated by western blot. * P<0.05 compared to the control group; # P>0.05 compared to the control group.
Figure 5.
The cell cycle distribution in>4N phase and endoreduplication were interrupted by treatment with a DNA poly β inhibitor or by DNA poly β knockdown.
(A) The expression of DNA poly β was examined by western blot as cells cultured with DDC or were transfected with siRNA DNA poly β. The results are presented as the ratio of the DNA poly β expression to β-actin expression. * P<0.05 compared to the control group; ** P<0.05 compared to the rotenone group; Rot: rotenone. (B) and (C) Cell cycle distribution and DNA replication were analyzed by flow cytometry using BrdU/PI staining after the addition of DDC or DNA poly β depletion. * P<0.05 compared to the rotenone group; # P>0.05 compared to the rotenone group; R3 represents endoreduplication; R2-R3 represents S phase; The data represent three independent experiments and are expressed as the mean ± SD.
Figure 6.
Expression of TH in the lesioned SN.
Brain tissue sections were cut to a thickness of 5 µm and were immunostained using an anti-TH antibody. The TH-positive neurons were visualized using a Cy3-conjugated donkey-anti-sheep IgG antibody. The TH-positive SN neurons were counted in the same coordinates of two coronal sections from the right brain of two rats. The bottom graph is the selective enlargement. * P<0.05 compared to the vehicle group; SNc: substantia nigra pars compacta; SNr: substantia nigra pars reticulate. Scale bar = 20 µm.
Figure 7.
After stereotactic (ST) infusion rotenone, the increased expression of cyclin D was induced in the SN neurons.
The immunoreactivity of cyclin D and TH was observed under the Laser confocal microscopy using DyLight 488-conjugated donkey-anti-rabbit IgG and Cy3-conjugated donkey-anti-sheep IgG, respectively, in the SN dopaminergic neurons of rats; The right graph is the selective enlargement; Scale bar = 20 µm.
Figure 8.
The expression of DNA poly β was enhanced in the lesioned brains of rats following rotenone infusion.
Double immunohistostaining of DNA poly β and TH was performed. The right part represents the selective enlargement. Scale bar = 20 µm.
Figure 9.
The selective increase of DNA poly β in the SN neurons in the ST-infused rats.
After rotenone infusion, the immunoreactivity of DNA poly β was detected. B-D show selective enlargements of A. Scale bar = 20 µm.