Fig. 4

The blockade to EnAd replication in murine cells is multifactorial. a NMuMG-CD46, CT26-CD46, and A549 cells were seeded at 1 × 10⁵ cells/well in a 24-well plate before infection with 5000 EnAd-CMV-GFP particles/cell or mock-infected. At 2 and 72 h post-infection, cells were washed and harvested for RNA extraction and subsequent cDNA conversion. Copy numbers of Ad11 (a) E1A, (b) E2B, and (c) Fiber mRNA in 50 ng cDNA were quantified by qPCR using Taqman probes. A synthetic oligonucleotide specific to the PCR product was used as a standard. “< 1” indicates conditions with values below 1 copy/50 ng DNA. d NMuMG, NMuMG-CD46, CT26, CT26-CD46, and A549 cells were seeded at 2 × 10⁵ cells/well in a 12-well plate before infection with 5000 EnAd-CMV-GFP particles/cell or mock-infected. At 2 and 72 h post-infection, cells were wash and harvested for genomic DNA extraction. EnAd genome copies in 30 ng of genomic DNA were quantified by qPCR using Taqman probes. e NMuMG, NMuMG-CD46, CT26, CT26-CD46, HCT116, and DLD1 cells were seeded at 1 × 10⁶ cells/well in a 6-well plate before infection with 5000 (5 K) or 1000 (1 K) EnAd-CMV-GFP particles/cell or mock-infected (M). At 72 h post-infection, cells were lysed and analysed for protein expression by immunoblotting. Late group B adenovirus structural proteins were visualised by a goat anti-adenovirus polyclonal antibody. Red text on the immunoblot are labels for 1, adenovirus hexon (105.3 kDa); 2, penton (62.3 kDa); and 3, fiber (36.1 kDa). Data represent biological triplicates, shown as mean ± SEM. Significance within each treatment was assessed using two-way ANOVA with Bonferroni’s Post Hoc analysis compared to infected A549 cells at 72 h post-infection (A-C) or to each corresponding measurement at 2 h post-infection (D). **, p < 0.01; ***, p < 0.001; ns, not significant. Black asterisks represent a significantly higher levels compared to infected A549 cells at 72 h post-infection; red, significantly lower levels