Figure 6

Effects of vimentin overexpression on NDV replication. A Confirmation of pcDNA3.1-VIM by restriction analysis. Vimentin fragment: 1380 bp; empty pcDNA3.1 vector: 5726 bp; marker: 1 kb DNA Ladder. B HD11 cells were transfected with a plasmid containing the full-length vimentin gene sequence (pcDNA3.1-VIM) or the empty plasmid (pcDNA3.1) for 24 h. The overexpression efficiency of vimentin was assessed by Western blotting using an anti-vimentin antibody. The band greyscale values were determined by ImageJ 1.4 software. Vimentin protein levels were normalized to ACTB protein levels and are shown relative to the levels in the Mock group. C HD11 cells were transfected with pcDNA3.1-VIM or pcDNA3.1 for 12 and 24 h. The transfected cells were harvested and used for a CCK-8 assay according to the manufacturer's instructions. The mock-transfected cells were assayed in parallel as a control. D HD11 cells were transfected with pcDNA3.1-VIM or pcDNA3.1 for 24 h and then infected with JS/7/05/Ch, JS/MukHN, or Mukteswar at an MOI of 1 for 24 h. The infected cells were harvested and lysed using RIPA buffer supplemented with the proteinase inhibitor PMSF. NDV NP expression levels were then measured by Western blotting with specific antibodies. The band greyscale values were determined by ImageJ 1.4 software. NP protein levels were normalized to ACTB protein levels and shown relative to pcDNA3.1. E HD11 cells were transfected with pcDNA3.1-VIM or pcDNA3.1 for 24 h and then infected with JS/7/05/Ch, JS/MukHN, or Mukteswar at an MOI of 1 for 6, 12, and 24 h. The supernatant of the infected cells was used for the determination of viral titres by a TCID50 assay. The statistical graphs were generated using GraphPad Prism 9.0 software. *p < 0.05; **p < 0.01; ***p < 0.001; ns indicates no significance.