Given the differences in gene expression and partial reversal of KCNJ6 downregulation, we evaluated detailed neuronal phenotypes in UN and AF iN cultures, including GIRK2 expression, neuronal morphology, and physiological response. Since GIRK2 immunocytochemistry had not been reported in cultured human neurons, we validated detection using mouse primary cortical cultures [42] (Fig. 2A, B). Lentiviral-transduced GIRK2 overexpression increased levels of immunostaining about 3.5-fold (Fig. 2C, mCherry+ cells are KCNJ6 lentiviral-transduced). Importantly, silencing KCNJ6 with shRNAs or frameshift knockout by CRISPR/Cas9 eliminated GIRK2 immunoreactivity (Supplementary Fig. 4B, C), which further confirms antibody specificity.
