Figure 3.

The detection of REST mRNA and protein in treated and untreated DS hiPSC-derived astrocytes and controls. Three pairs of cell lines (C2 vs. DS2, C4 vs. DS3, and C5 vs. DS4) were used to perform ICC. (A) The mRNA expression levels of REST in different groups: Con group (Control; Disomic astrocytes), Con + X5050 group (Disomic astrocytes with X5050 treatment), DS + Li group (Trisomic astrocytes with lithium carbonate treatment), DS + Li + X5050 (Trisomic astrocytes treated with both lithium carbonate and X5050). The mRNA expression levels were normalised to two reference genes (HMBS and PSMB2), and the relative mRNA expression levels for each group were calculated. The relative expression levels of each group were normalised to those of the Con group to obtain log2 fold changes for statistical analyses. (B) Nucleus REST expression analysis based on the (C) fluorescence micrographs. (D) Optimisation of safety dosage of lithium treatment in iPSC-derived astrocytes through MTT cell viability assay. Treatment with lithium carbonate up to 5 mM for 24 h is considered safe for astrocytes in which the cell viability does not drop below 100%. (E) Optimising safe doses of lithium for iPSC-derived astrocytes via MTT cell viability assay. Cell viability treated with 100 μM X5050 for 24 h was not statistically significant compared to the control and was considered safe. The one-way ANOVA was employed to assess the differences between groups. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001. Scale bar = 50 μm.