PMC

Basal levels of GSH in microglia and astrocytes. GSH levels were measured by HPLC. Basal GSH levels in astrocytes (93.43±16.62nmol/mg protein) were approximately 4-fold higher compared to microglia (26.71±3.85 nmol/mg protein). Values are expressed as the mean ± SEM derived from three independent experiments. ★ P<0.05, ★ ★ P<0.01, ★ ★ ★ P<0.001

LDH analysis of MeHg-treated astrocytes and microglia. The effects of MeHg cytotoxicity were measured with the LDH assay after MeHg treatment for 6 hr. Values are expressed as the mean ± SEM derived from three independent experiments. ★ P<0.05, ★ ★ P<0.01, ★ ★ ★ P<0.001

MTT analysis of MeHg-treated astrocytes and microglia. The effects of MeHg cytotoxicity were measured with the MTT assay after MeHg treatment for 6 hr. Values are expressed as the mean ± SEM derived from three independent experiments. ★ P<0.05, ★ ★ P<0.01, ★ ★ ★ P<0.001

ROS production in astrocytes and microglia. DCF fluorescence was measured at 1 min, 10 min and 1 hr after MeHg treatment both in microglia and astrocytes. MeHg effects at 6 hr were collected only in astrocytes. Values are expressed as the mean ± SEM derived from six independent experiments. ★ P<0.05, ★ ★ P<0.01, ★ ★ ★ P<0.001

Nrf2 protein level in whole cell lysates of microglia and astrocytes. The Nrf2 protein level in whole cell lysates was measured by western blot analysis. Both cell types were treated with MeHg for 1 min, 10 min and 1 hr. Astrocytes were also treated with MeHg for 6 hr. Values are expressed as the mean ± SEM derived from three independent experiments. ★ P<0.05, ★ ★ P<0.01, ★ ★ ★ P<0.001

Expression of Ho1, Nqo1 and xCT in microglia and astrocytes. The gene expression level was measured by real-time PCR. The differences in the average threshold cycle (ΔCt) values were determined and normalized to the expression of β-actin. (A) Microglia were treated with MeHg for 30 min. (B) Astrocytes were treated with MeHg for 6 hr. Values are expressed as the mean ± SEM derived from three independent experiments. ★ P<0.05, ★ ★ P<0.01, ★ ★ ★ P<0.001

GSH/GSSG ratios in microglia and astrocytes. GSH and GSSG concentrations were measured by HPLC, and the ratios of GSH/GSSG were calculated. The ratio of the control group was set to 100%. Both cell types were treated with MeHg for 1 min, 10 min and 1 hr. Astrocytes were also treated with MeHg for 6 hr. Values are expressed as the mean ± SEM derived from three independent experiments. ★ P<0.05, ★ ★ P<0.01, ★ ★ ★ P<0.001

The effect of Nrf2 knockdown on cell viability in microglia and astrocytes. Cell viability was assessed with the MTT assay. Both cell types were first infected for 24 hr with lentivirus and then treated with 5 μM MeHg for 6 hr. The uninfected cells in the absence of MeHg treatment were used to determine the maximal cell viability. Values are expressed as the mean ± SEM derived from three independent experiments. ★ P<0.05, ★ ★ P<0.01, ★ ★ ★ P<0.001

Nrf2 immunostaining in microglia and astrocytes. Nrf2 was labeled in green, cell nuclei were stained with PI dye in red, and the yellow coloring reflects the colocalization of Nrf2 and DNA in microglial nuclei. (A) In microglia, controls showed minimal Nrf2 protein expression in the cytosol. Treatment with 0.1 μM of MeHg for 10 min increased Nrf2 in the cytosol, but not in the nuclei. Higher concentrations of MeHg increased Nrf2 in both the cytosol and nuclei. (B) In astrocytes, treatment with only 5 μM MeHg for 6 hr increased Nrf2 protein expression in both the cytosol and nuclei. Photographs show representative fields observed from four independent experiments. ★ P<0.05, ★ ★ P<0.01, ★ ★ ★ P<0.001

Intracellular mercury (Hg) concentration in microglia and astrocytes. Both cell types were treated with a mixture of ¹⁴C-MeHg and non-radioactive MeHg (volume ratio of 1:1000) at for 1 min, 10 min, 1 hr and 6 hr. The radioactivity was measured, and intracellular Hg levels were calculated and normalized to the protein concentrations. Notably, astrocytes had consistently lower intracellular Hg levels compared to microglia. Values are expressed as the mean ± SEM derived from three independent experiments. ★ P<0.05, ★ ★ P<0.01, ★ ★ ★ P<0.001

The effect of Nrf2 knockdown on Ho1, Nqo1 and xCT expression in MeHg-treated microglia and astrocytes. Both cell types were infected with lentivirus for 24 hr. Next, 5 μM MeHg was added for 30 min in microglia (A) and for 6 hr in astrocytes (B). Uninfected cells in the absence of MeHg treatment were used to measure the basal gene expression level. The mRNA levels of Ho1, Nqo1 and xCT were measured by real-time PCR. The difference in the average threshold cycle (ΔCt) value was determined and normalized to the expression of β-actin. Values are expressed as the mean ± SEM derived from three independent experiments. ★ P<0.05, ★ ★ P<0.01, ★ ★ ★ P<0.001