Small interfering RNA (siRNA)-mediated knockdown of zinc finger binding protein 804A (ZNF804A) in CTX0E16 neurons. (A) Representative images of ZNF804A staining and neurite length in early stage (DD7) CTX0E16 neurons after a 7-day treatment with no transfection (blank), control (scramble), or ZNF804A-targeting siRNAs. (B) Total neurite length was significantly reduced in neurons treated with both siRNAs relative to the scramble siRNA control (n = 3 independent experiments). (C, D) Western blot analysis of synaptic and adhesion protein expression in control and siRNA expressing CTX0E16 neurons. In ZNF804A knockdown cells, neuroligin-4 (NLGN4) is significantly reduced; N-cadherin and AMPA receptor subunit 2 (GluA2) are unaffected. (E) Representative images of early stage (DD7) CTX0E16 neurons after a 7-day treatment with no transfection (blank), control (scramble), or ZNF804A-targeting siRNAs, with or without exogenous expression of HA-NLGN4 for 2 days. (F) Quantification of neurite outgrowth of cells in panel E. siRNA-treated cells had significantly reduced outgrowth compared with blank or scramble conditions. Neurons treated with ZNF804A-siRNA and HA-NLGN4 had significantly longer neurites than siRNA alone conditions, but they did not differ from control conditions (n = 3–4 independent experiments). Scale bars = 10 µm (A1), 50 µm (A2), and 20 µm (E). AMPA, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; a.u., arbitrary unit; DAPI, 4′,6-diamidino-2-phenylindole; N-cad, N-cadherin. *p < .05, **p < .01.