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. 2021 Mar 10;41(10):2287–2300. doi: 10.1523/JNEUROSCI.3018-19.2021

Figure 4.

Figure 4.

Gemfibrozil upregulates the expression of Gdnf via PPARα−/− in mouse primary astrocytes. Primary astrocytes isolated from 2- to 3-d-old pups from both genotypes were treated with increasing concentrations of gemfibrozil (µm) for 2 h under serum-free conditions followed real-time PCR analysis to assess Gdnf mRNA expression. A, WT astrocytes. Results are the mean ± SD of three independent experiments, and the significance of the mean was tested by one-way ANOVA (F(5,12) = 4.916 > Fc = 3.68; *p = 0.0112). A post hoc Tukey's test results in *p = 0.015 for 10 μm gemfibrozil versus control, and **p = 0.002 for 15 μm gemfibrozil versus control. B, PPARα−/− astrocytes. Results are the mean ± SD of three independent experiments. Significance of the mean was tested by one-way ANOVA but found no significant differences among the means (F(5,12) = 0.8125 < Fc = 3.68; p = 0.562). C, D, Cells were treated with gemfibrozil for 18 h under serum-free conditions followed by monitoring GDNF in WT astrocytes by immunoblotting (C) that was quantified via densitometric analyses (D). Results are the mean ± the SEM of three independent immunoblots. Significance of the mean tested by one-way ANOVA (F(5,12) = 24.59 > Fc = 3.68). A post hoc Tukey's test results in **p = 0.0049 for 10 μm gemfibrozil versus control for 15 μm gemfibrozil versus control. E, F, GDNF expression in PPARα−/− astrocytes was visualized by immunoblotting (E) and quantified via densitometric analyses (F). Results are the mean ± SD of three independent immunoblots and the significance of the mean was tested by one-way ANOVA (F(5,12) = 4.695 > Fc = 3.68; *p = 0.0132). A post hoc Tukey's test was used to find significant differences among the treatment groups and shows a significant reduction of GDNF protein compared with control at doses of 15 μm in PPARα−/− primary astrocytes. G, Mouse primary astrocytes were infected with GFP-control lentivirus and mouse GDNF sgRNA CRISPR all-in-one lentivirus (6 × 106 IU/ml) according to manufacturer protocol and 2 d after infection, the level of GDNF protein was monitored in cells by Western blot. Actin was run as a loading control. Results represent CRISPR editing of three different wells. H, I, Protein expression levels were corroborated by double labeling WT (H) and PPARα−/− (I) astrocytes with antibodies to GDNF (cy-5) and GFAP (cy-3). DAPI was used to visualize the nucleus. Scale bar, 20 µm.