HCMV IE1 is sufficient and necessary to reduce Hes1 protein levels.

(A) Effect of IE1, IE2 and pp65 expression on endogenous Hes1 protein levels in NPCs. At 48h following nucleofection with 5μg pCDH-GFP (vector), pCDH-IE1 (IE1), pCDH-IE2 (IE2) or pCDH-pp65 (pp65), NPCs were collected and subjected to IB for Hes1. (B) Effect of IE1 deletion on Hes1 protein downregulation during infection in NPCs. NPCs were mock-infected (M) or infected with TNwt (wt), TNdlIE1 (dlIE1) or TNrvIE1 (rvIE1) at an MOI of 10. Cells collected at 12hpi were subjected to IB. (C) Effect of IE1 knock-down on protein level of endogenous Hes1 during HCMV infection. At 48 h following transduction with lentiviruses expressing a scrambled shRNA (Scram) or an shRNA targeting IE1 (sh-IE1), NPCs were infected with HCMV (MOI = 1) for 24h and subjected to IB for Hes1 and IE1. (D) Effect of IE1 expression on protein levels of exogenous Hes1. At 48h following co-transfection with the indicated amount of pCDH-Hes1 (Hes1) and pEYFP-GFP (vector) or pEYFP-IE1 (IE1), 293T cells were collected and subjected to IB for Hes1. The values listed below the blots indicate the relative Hes1 protein levels compared to the corresponding controls following β-actin normalization. Representative images from 3 independent experiments are shown (left), and the relative levels of Hes1 are presented as the mean ± SD (right). *, P≤0.05; **, P≤0.01.