Fig. 2

Neuronal upregulation of ISG15 in vitro in response to interferon treatment. Cortical neurons prepared from E15.5 embryonic mouse cortices (A) and human IPSC-derived neurons (B) were plated into microfluidic axon isolation chambers. After 10 days, axons exhibited dense outgrowth into the distal chamber. At DIV 12, 100 ng/mL IFNγ or PBS was added to the distal chamber, and 72 h following axonal treatment with IFNγ or PBS gene expression was assessed by microarray. C At DIV 12, neurons were treated for 24 h with 2000 U/mL IFNα, 100 ng/mL IFNγ or 2 ug/mL PolyI:C to drive ISG15 expression. Lysates were probed for ISG15 (F9; SantaCruz) and acquired on film with Xomat film processor. Membranes were stripped and reprobed for B-actin as loading control. Blots show high molecular weight smear indicative of ISGylated targets and bands at 17kd indicative of ISG15 monomer. Murine cortical neurons (D) were transfected with AAV1.Syn.EGFP with or without co-transfection with AAV1.Syn.shISG15 to silence neuronal ISG15 expression and 12 days later were treated for 24 h as in panel C and then fixed and immunostained for ISG15. Representative confocal images show ISG15 expression (red) in IFN-treated neurons (green) as well as non-neuronal cells. Neuronal ISG15 expression was silenced by AAV1.Syn.shISG15 co-transfection. Human IPSC-derived neuronal aggregates (E) and murine cortical neurons cultures (F) were cultured in microfluidic chambers for 12 days and then IFNγ (100 ng/mL) was added to either the axon or soma compartment of the microfluidic chamber. RNA was collected 1–48 h later as indicated and expression levels of the indicated genes were determined by RT-PCR relative to control neurons. Mean ± SEM are shown