Fig 1.
The kinetics of thymocytes apoptosis at different days post-infection (dpi).
The percentages of apoptotic cells in the thymus of one representative HuN4-infected piglet at 7 (A) and 10 (C) dpi, and apoptotic cells in the thymus of a representative age-matched control piglet at 7 (B) and 10 (D) dpi. Apoptotic cells were quantified by flow cytometry using FITC-labeled Annexin V. Q1, necrotic or another cell population that was FITC-Annexin V-negative and PI-positive; Q2, end stage apoptotic or a dead cell population that was FITC-Annexin V- and PI-positive; Q3, a viable cell population that was not undergoing apoptosis and was both FITC-Annexin-V- and PI-negative; Q4, an early apoptotic cell population that was FITC-Annexin V-positive and PI-negative.
Fig 2.
Apoptosis and autophagy are induced by HuN4 infection in the thymus of infected piglets.
Levels of proteins related to apoptosis or autophagy in the thymus of piglets at 7 (A) and 10 (B) dpi. Thymus tissues were lysed and western blots were performed. Western blotting results were analyzed digitally and the optical density ratio was calculated, and the bands which were in use for densitometry marked by the arrows; the ratio representatives the mean of measurements for piglets at different time points and the Student’s t-test were carried for the statistical analysis. (n = 3 per time point).
Fig 3.
Colocalization of PRRSV-infected cells, autophagic cells, and nuclei.
Autophagic cells were stained with anti-LC3 antibodies and anti-rabbit secondary antibodies conjugated to FITC, and apoptotic cells were detected according to the manufacturer’s instructions for the In Situ Cell Death Detection Kit, only a few apoptotic cells underwent autophagy (arrows) (A); Autophagic cells were stained with anti-LC3 antibodies and tetramethylrhodamine- isothiocyanate (TRITC)-conjugated goat anti-mouse antibodies, and HuN4-infected cells were strained with fluorescein isothiocyanate (FITC)-conjugated monoclonal antibodies (mAbs) against PRRSV N protein, and some HuN4-infected cells underwent autophagy (arrows) (B). Nuclei were stained with 4-6-diamidino-2-phenylindole (DAPI).
Fig 4.
Colocalization of CD3+ cells, CD14+ cells, thymic epithelial cells, and autophagic cells.
CD3+ cells were stained with spectral red (SPRD)-conjugated mouse anti-pig CD3 antibodies, and autophagic cells were stained with anti-LC3 primary antibodies and anti-rabbit secondary antibodies conjugated to FITC, no CD3+ cells underwent autophagy (A); CD14+ cells were stained with FITC-conjugated mouse anti-pig CD14 antibodies and autophagic cells were stained with anti-LC3 primary antibodies and TRITC-conjugated goat anti-mouse antibodies, some CD14+ cells underwent autophagy (arrows) (B). Thymic epithelial cells were stained with mouse anti-pan-cytokeratin (P-CK) antibodies and FITC-conjugated goat anti-mouse antibodies, and autophagic cells were stained with anti-LC3 primary antibodies and TRITC-conjugated goat anti-mouse antibodies, most autophagic cells colocalized with thymic epithelial cells (arrows) (C). Nuclei were stained with DAPI.