PRRSV infection triggers mitochondrial fission and mitophagy mediated by mitochondrial Ca<sup>2+</sup> in primary cells.
<p>(A-C) PAMs were mock-infected or PRRSV-infected (MOI = 0.1) for 18 h. (A) The cells were stained with MitoTracker Red, PRRSV-N antibody and DAPI, followed by confocal microscopy. Quantitative analysis of mitochondrial length is presented on the right. The lengths of mitochondria from 20 cel...
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2025
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| Summary: | <p>(A-C) PAMs were mock-infected or PRRSV-infected (MOI = 0.1) for 18 h. (A) The cells were stained with MitoTracker Red, PRRSV-N antibody and DAPI, followed by confocal microscopy. Quantitative analysis of mitochondrial length is presented on the right. The lengths of mitochondria from 20 cells were quantified for each group. (B) Cell lysates were harvested for western blot analysis. (C) Detection of mitochondrial Ca<sup>2+</sup> using Rhod-2 staining by flow cytometry. (D and E) PAMs were exposed to PRRSV (MOI = 0.1) for 18 hours, either with or without Mdivi-1 (2.5 μM) treatment. (D) Western blot analysis was performed on the cell lysates by using antibodies against DRP1, PRRSV-N and β-actin. (E) Assessment of the TCID<sub>50</sub> of PRRSV in the cell supernatant. (F and G) PAMs were either treated or not treated with VBIT-12 (5 μM), then mock-infected or infected with PRRSV (MOI = 0.1) for 18 hours. (F) Ca<sup>2+</sup> levels were assessed using flow cytometry with Rhod-2 staining. (G) The cell lysates underwent western blot analysis using antibodies specific to DRP1 Ser637, DRP1 Ser616, DRP1, PRRSV-N and β-actin. (H) PAMs were mock-infected or infected with PRRSV (MOI = 0.1) for 18 hours. Collect cell lysates and perform western blot analysis using antibodies against PINK1, Parkin, LC3, PRRSV-N, and β-actin. (I-L) PAMs cells were transfected with siRNA targeting PINK1 (I and J) or Parkin (K and L) for 24 hours, followed by 18 hours of mock or PRRSV infection (MOI = 0.1). (I and K) Cell lysates were harvested for western blot analysis with antibodies against PINK1 or Parkin, PRRSV-N and β-actin. (J and L) Determination of the TCID<sub>50</sub> of PRRSV in the cell supernatant. (M and N) PAMs were either treated or not treated with VBIT-12 (5 μM), then mock-infected or infected with PRRSV (MOI = 0.1) for 18 hours. (M) Cell lysates were harvested for western blot analysis with antibodies against PINK1, Parkin, LC3, PRRSV-N, and β-actin. (N) Determination of the TCID<sub>50</sub> of PRRSV in the cell supernatant. Data are expressed as means ± SD, n = 20 in A or n = 3 in C, E, F, J, L, N. *p<0.05; **p < 0.01; ***p < 0.001. The levels of phosphorylated DRP1 were normalized to the total DRP1 protein, while the levels of other proteins were normalized to β-actin. The data are representative of results from three independent experiments.</p> |
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