Figure 4.

Effects of pharmacologic and genetic manipulation of $\text{HIF-}1\mathrm{\alpha }$ on cell proliferation. (A) Astrocytes were pretreated with ${\text{CoCl}}_2$ ($200\mathrm{\mu }\mathrm{M}$, $0.5\mathrm{h}$) and then treated with $10\mathrm{\mu }\mathrm{M}$ MeHg for $0.5\mathrm{h}$. Western blotting was used to evaluate protein levels of $\text{HIF-}1\mathrm{\alpha }$, GLUT-1, EPO, and VEGF-A. (B) Effect of ${\text{CoCl}}_2$ pretreatment ($200\mathrm{\mu }\mathrm{M}$, $0.5\mathrm{h}$) on the cell proliferation was evaluated using an MTT assay. (C) Effect of 2-MeOE2 pretreatment ($10\mathrm{\mu }\mathrm{M}$, $0.5\mathrm{h}$) on the expression of $\text{HIF-}1\mathrm{\alpha }$, GLUT-1, EPO, and VEGF-A was detected by Western blotting. (D) Effect of 2-MeOE2 pretreatment ($10\mathrm{\mu }\mathrm{M}$, $0.5\mathrm{h}$) on the cell proliferation was evaluated using an MTT assay. For Western blotting analyses, representative blots are shown, and the intensities are presented as fold changes relative to the control group ($\mathrm{\beta }\text{-actin}$ as the internal control). *$p<0.05$ vs. control, ^#$p<0.05$ vs. MeHg-only group. (E) The protein expression levels of $\text{HIF-}1\mathrm{\alpha }$, GLUT-1, EPO, and VEGF-A after overexpression of $\text{HIF-}1\mathrm{\alpha }$. *$p<0.05$ vs. HBAD-null group. (F) Effect of adenovirus-induced $\text{HIF-}1\mathrm{\alpha }$ overexpression on the decrease in MeHg-induced cell proliferation as determined by an MTT assay. *$p<0.05$ vs. control group, ^#$p<0.05$ vs. $\text{MeHg}+\text{HBAD-null}$ group. (G) Effect of $\text{HIF-}1\mathrm{\alpha }$ siRNA on the protein expression of $\text{HIF-}1\mathrm{\alpha }$, GLUT-1, EPO, and VEGF-A. (H) Effect of $\text{HIF-}1\mathrm{\alpha }$ siRNA on the MeHg-induced decrease in cell proliferation. Note: Statistical analysis was performed by one-way ANOVA with Tukey’s post hoc test. Data are presented as the $\text{mean}\pm \text{SD}$ from three independent experiments ($n=3$). ${\text{CoCl}}_2$, cobalt chloride; con, control (culture medium treatment without MeHg); EPO, erythropoietin; GLUT-1, glucose transporter 1; HBAD, pHBAd-EF1-MCS-GFP; $\text{HIF-}1\mathrm{\alpha }$, Hypoxia-inducible $\text{factor-}1\mathrm{\alpha }$; 2-MeOE2, 2-methoxyestradiol; MeHg, methylmercury; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl diphenyltetrazolium bromide; nc siRNA, negative control (scrambled sequence) siRNA; siRNA, small interfering RNA; VEGF-A, vascular endothelial growth factor A. *$p<0.05$ vs. nc siRNA group. *$p<0.05$ vs. control group, ^#$p<0.05$ vs. $\text{MeHg}+\text{nc \hspace{0.17em} siRNA}$ group.