Figure 1.
Rotenone directly activated the NF-κB signaling pathway in BV2 cells.
(A) Various doses of rotenone or LPS were administered to BV2 cells as indicated for 6 hours or 1 hour, respectively. The cell lysates were immunoblotted with anti-pIKK or anti-IKK antibodies. GAPDH served as the loading control. (B) BV2 cells were treated as described in (A). The cell lysates were immunoblotted with anti-IκB antibodies. (C) Quantitative analysis of the data from (B), showing the density of IκB relative to that of the loading control (GAPDH). The data are presented as the mean ± S.E.M. n = 3, *p<0.05, one-way ANOVA. (D) BV2 cells were treated with rotenone for 6 hours or LPS for 1 hour. The cells were fixed and labeled with DAPI (blue) or anti-p65 antibodies (red). The scale bar represents 10 µm. (E) The cytoplasmic and nuclear fractions from the treated BV2 cells were immunoblotted with anti-p65, anti-GAPDH or anti-Max antibodies. (F) Quantitative analysis of the data from (E), showing the density of nuclear p65 relative to that of the loading control (Max). The data are presented as the mean ± S.E.M. n = 3, *p<0.05, **p<0.01, one-way ANOVA.
Figure 2.
Rotenone induced inflammatory cytokine expression in BV2 cells.
(A) BV2 cells were treated with different doses of rotenone or LPS for 24 hours. The TNFα levels in the cultured media were measured using ELISA assays. The data are presented as the mean ± S.E.M. n = 3, **p<0.01, one-way ANOVA. (B) Immunoblot analysis to detect iNOS expression in the BV2 cells treated with rotenone or LPS. (C) Quantitative analysis of the data from (B), showing the density of iNOS relative to that of the loading control (tubulin). The data are presented as the mean ± S.E.M. n = 3, **p<0.01, ***p<0.001, one-way ANOVA.
Figure 3.
Rotenone induced inflammasome activation in BV2 cells.
(A) BV2 cells were treated with 1 µM rotenone or 1 µg/mL LPS for 24 hours and then stained with green fluorescent probes for activated caspase-1 for 1 hour. The scale bar represents 10 µm. (B) BV2 cells were treated with different doses rotenone or LPS, as indicated for 24 hours. The cell lysates were immunoblotted using anti-caspase-1 antibodies. The long-exposure film showed the cleaved caspase-1 (the active p10 form) in the BV2 cells treated with the higher doses of rotenone or LPS. The total caspase-1 levels are shown in the short-exposure film. (C) BV2 cells were treated in the same manner as those in (B). The cell lysates were immunoblotted with anti-IL-1β antibodies. The activated IL-1β cleaved by caspase-1 is indicated as cleaved IL-1β.
Figure 4.
Rotenone induced microglial activation via p38 MAPK.
(A) BV2 cells were treated with different doses of rotenone for 6 hours or 1 µg/mL LPS for 1 hour and lysed in lysis buffer. The lysates were immunoblotted with the indicated antibodies. (B) Quantitative analysis of the data from (A), showing the density of pp38 relative to that of the loading control (tubulin). The data are presented as the mean ± S.E.M. n = 3, *p<0.05, one-way ANOVA. (C) BV2 cells were treated with 1 µM rotenone for 6 hours or 1 µg/mL LPS for 1 hour or co-treated with 5 mM NAC and either 1 µM rotenone for 6 hours or 1 µg/mL LPS for 1 hour. The cells were stained with an ROS indicator, DCFH-DA, for 30 minutes. The scale bar represents 10 µm. (D) BV2 cells were treated with rotenone or LPS in the same manner described in (C). The cells were lysed, and the lysates were immunoblotted with the indicated antibodies.
Figure 5.
NAC blocked activation of the rotenone-induced NF-κB signaling pathway.
(A) BV2 cells treated with 1 µM rotenone for 6 hours or 1 µg/mL LPS for 1 hour or co-treated with 5 mM NAC and either 1 µM rotenone for 6 hours or 1 µg/mL LPS for 1 hour. The cells were immunoblotted with anti-IκB antibodies. Tubulin served as the loading control. (B) BV2 cells treated with 1 µM rotenone for 6 hours or 1 µg/mL LPS for 1 hour or co-treated with 5 mM NAC and either 1 µM rotenone for 6 hours or 1 µg/mL LPS for 1 hour. The cells were fixed and labeled with DAPI (blue) and anti-p65 antibodies (red). The scale bar represents 10 µm. (C) The cytoplasmic and nuclear fractions from the BV2 cells that were treated as indicated in (B) were subjected to immunoblot analysis with anti-p65 antibodies. GAPDH served as the marker for the cytoplasmic fraction, and Max served as the marker for the nuclear fraction.
Figure 6.
NAC blocked the rotenone-induced microglial activation.
(A) BV2 cells treated with 1 µM rotenone for 24 hours or 1 µg/mL LPS for 24 hours or co-treated with 5 mM NAC and either 1 µM rotenone for 24 hours or 1 µg/mL LPS for 24 hours. The TNFα levels in the culture media of the treated BV2 cells were measured by ELISA. (B) BV2 cells were treated with as indicated in (A). Then, the cells were lysed, and the lysates were immunoblotted with the indicated antibodies. The activated IL-1β cleaved by caspase-1 is indicated as cleaved IL-1β.