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. 2024 Oct 1;5(10):101751. doi: 10.1016/j.xcrm.2024.101751

Figure 6.

Figure 6

Construction and characterization of the recombinant adenovirus ADVTα1

(A) Schematic diagram of a recombinant adenovirus expressing Tα1, ΔE1 contains E1B55K-deletion. The expression of EGFP and Tα1 is driven by inserting CMV and EF-1α promoters in the E1 region, respectively.

(B) 4T1 cells were infected with ADV or ADVTα1 (MOI = 2) for 48 h, and the secretion of Tα1 was confirmed by western blotting.

(C) 4T1 cells and MDA-MB-231 cells were infected with ADVTα1 or ADV at different MOIs, and cell viability was measured 48 h later by CCK-8 assay (n = 3 biological replicates).

(D) 4T1 cells and MDA-MB-231 cells were infected with ADVTα1 or ADV at an MOI of 1. The virus titers were measured by TCID50 assay at different time points after infection (n = 3 biological replicates).

(E) Infection of 4T1 cells with ADV or ADVTα1 (MOI = 10) for 24 h. Early apoptotic cells were confirmed as Annexin-V+/PI− cells, and late apoptotic (and/or necrotic) cells were confirmed as Annexin-V+PI+ cells (n = 3 biological replicates).

(F) 4T1 cells, CT26 cells, H22 cells, MDA-MB-231 cells, HCT116 cells, HepG2 cells, and MET-5A cells were infected with ADVTα1 or ADV at an MOI of 1. The expression of EGFP in the cells was measured by FCM.

(G–K) Macrophages (THP-1 cells) were cocultured with 231ADV (ADV), 231ADV cells with Tα1 (ADV + Tα1), or ADVTα1-infected MDA-MB-231 cells (ADVTα1), followed by analysis of macrophage polarization. (F) Schematic representation of experimental design. (H) Expression of CD86 (top) and CD206 (bottom) in macrophages was measured by FCM (n = 3 biological replicates). (I) Expression of Ki67 was measured by FCM in CD86+ macrophages or CD206+ macrophages (n = 3 biological replicates). (J) Expression of iNOS, IL-1β, IL-6, Arg-1, and IL-10 in macrophages was measured by qPCR (n = 3 biological replicates and 3 technical replicates). (K) The mRNA expression level of iNOS, IL-1β, IL-6, Arg-1, and IL-10 (from Figure 6J) was showed as heatmap.

The data are shown as the means ± SD. NS, no significant difference; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.