Analysis on the expression of viral and cellular gene products in NSPC/iPSCs. (A) Morphological changes of Towne-infected NSPC/iPSCs were observed under the inverted microscope before infection (a), 2 dpi (b), 5 dpi (c), and 7 dpi (d). (B) RT-PCR analysis of HCMV-encoding gene expression. Total RNAs isolated from NSPC/iPSCs harvested before (−) HCMV infection or at 1, 2, 3, 5, and 7 dpi with HCMV Towne strain were subjected to RT-PCR assays. GAPDH gene expression was assayed for the control. (C) The kinetics of mRNA expression for IE1, UL89, and UL136 in Towne-infected NSPC/iPSCs was examined by real-time quantitative RT-PCR assay. The mRNA expression was normalized to that of G6PDH gene. Real-time PCR data was analyzed by the 2-ΔΔCT method. The fold induction was calculated as the ratio of mRNA levels detected at each time point to that detected at 1 dpi. The y-axis represents fold induction of IE1 and UL136 mRNA (left y-axis) and UL89 mRNA (right y-axis). (D) Immunoblot analysis of HCMV protein expression in HCMV-infected NSPC/iPSCs. Whole-cell lysates of NSPC/iPSCs harvested before (−) HCMV infection or at 1, 2, 5, and 7 dpi with HCMV Towne strain were separated by SDS-PAGE and analyzed by immunoblotting with antibodies against IE1/IE2, pp65, gB, and α-tubulin. (E) RT-PCR analysis of pluripotency and neural differentiation marker gene expression in HCMV-infected NSPC/iPSCs. (F) Immunoblot analysis of neural differentiation marker protein expression in HCMV-infected NSPC/iPSCs. Whole-cell lysates of NSPC/iPSCs were analyzed by immunoblotting with antibodies against Musashi-1, Pax6, and Nestin. (G) hTERT-BJ1 cells inoculated with culture supernatant collected from mock-infected NSPC/iPSCs (upper panel) or Towne HCMV-infected NSPC/iPSCs (lower panel) at 8 dpi were subjected to immunofluorescence test with anti-IE1/IE2 antibody (green). Nuclei were stained with DAPI. Representative results from two independent experiments are shown.