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. 2015 Aug 19;89(21):10912–10923. doi: 10.1128/JVI.01589-15

FIG 3.

FIG 3

Effects of inhibition of the ERK/MAPK and PI3K signaling pathways on EHV-1-induced CD172a+ cell adhesion to EC monolayers. (A) Western blot analyses of the levels of phosphospecific ERK1/2 and total ERK1/2, as well as the levels of phosphospecific Akt and total Akt, between mock- and EHV-1-inoculated CD172a+ cells in the presence or absence of ERK/MAPK and PI3K inhibitors. One million CD172a+ cells were preincubated with the MEK/ERK inhibitor U0126 (10 μM), the PI3K inhibitor LY294002 (50 μM), or DMSO-based diluent for 30 min at 37°C. Cells were (mock) inoculated with EHV-1 strain 03P37 or 97P70 (MOI = 1) and lysed at 0, 5, and 15 min postinoculation. As a control for loading, total β-actin levels were assessed. (B and C) CD172a+ cells were preincubated with U0126 (1 or 10 μM) (B) or LY294002 (25 or 50 μM) (C) for 30 min at 37°C. The cells were (mock) inoculated with EHV-1 strain 03P37 or 97P70 (MOI = 1) and cocultured for 2 h with immortalized EC. The number of adherent CD172a+ cells per square millimeter was calculated. (D) Low-magnification confocal images of the reduced adhesion of blood CD172a+ cells to EC upon inhibition of the ERK/MAPK signaling pathway. Nuclei were counterstained with Hoechst (blue). The arrow points to an adhering monocytic cell. All the confocal images shown represent merged z-stacks. Scale bars, 100 μm. (E) U0126 or LY294002 pretreatment did not decrease EHV-1 infection of CD172a+ cells. CD172a+ cells adherent to plastic were pretreated with U0126 (10 μM), LY294002 (25 μM), or DMSO-based diluent for 30 min at 37°C and inoculated with EHV-1 strain 03P37 or 97P70 (MOI = 1) for 1 h at 37°C in LM supplemented with inhibitors. The cells were further incubated in LM for 12 h at 37°C. The percentage of IEP-positive cells was calculated based on 300 cells counted in distinct fields. Experiments were performed three times. The error bars show the SD, and a two-way ANOVA test was performed to evaluate significant differences from controls (ns, not significant; *, P ≤ 0.001; **, P < 0.0001).