FIG 4.

PRRSV induces macropinocytosis via TIM-1 in MARC-145 cells. (A) Knockdown of TIM-1 greatly influenced macropinocytosis. After transfection with siRNA-NC or siTIM-1 for 36 h, PRRSV BJ-4 (MOI = 10) was applied to the serum-starved MARC-145 cells at 4°C. The input virus was replaced with medium containing dextran (final concentration, 250 μg/ml) and transferrin (final concentration, 10 μg/ml) and transferred to 37°C for 30 min. The cells were then fixed, and the nuclei were stained with DAPI. Images were acquired with the same confocal microscope settings. The total fluorescence intensity of transferrin or dextran was calculated using ImageJ software. ***, P < 0.001; ns, not significant. (B) PRRSV induced macropinocytosis via TIM-1. MARC-145 cells were transfected with mock treatment, siRNA-NC, or siTIM-1 for 36 h and then serum starved for 2 h. The cells were inoculated with or without PRRSV BJ-4 (MOI = 10) at 4°C. The inoculum was replaced with medium containing FITC-dextran and transferred to 37°C for 15, 30, or 45 min. The cells were fixed and examined by confocal microscopy using the same confocal microscope settings. (C) The total fluorescence intensity of the dextran in panel B was calculated using ImageJ software. **, P < 0.01; ***, P < 0.001. (D) Serum-starved and siRNA-transfected MARC-145 cells were inoculated with PRRSV BJ-4 (MOI = 10) for 30 min, followed by dextran uptake (green). PRRSV infection is indicated by anti-PRRSV N protein antibody (red). White arrows indicate the colocalization of dextran and PRRSV. (E) PRRSV induced membrane protrusions. The serum-starved MARC-145 cells were added with PRRSV BJ-4 (MOI = 10) or PBS for 30 min and fixed with 4% PFA. Actin filaments were labeled with phalloidin (green). Images were captured with a 63× oil immersion objective. A higher magnification of the boxed area shows the formation of actin protrusions on the cell surface (white arrows). Scale bars, 10 μm.