Figure 4.
SINGER-mediated transgene expression in a tumor xenograft mouse model
(A) Schematic illustration of the experimental design for evaluating SINGER-mediated transgene expression in tumors.
(B and C) Time-course-dependent SINGER-mediated transgene expression kinetics in tumors revealed by bioluminescence images in mice. C57BL/6 mice bearing B16F10 melanoma tumors received an intratumoral injection of VNP20009 cells (5 × 105 colony-forming units [CFUs]) transformed with pGT240, after which the mice were exposed to US (0.5 W/cm2, pulse with 1 s on, 1 s off) for the indicated durations. The bioluminescence signal intensity was quantified with an in vivo imaging system 12 h after US irradiation. The data in (C) are presented as means ± SEM; n = 4 mice. p values were calculated by one-way ANOVA with Tukey’s post-test. ∗∗p < 0.01 and ∗∗∗p < 0.001.
(D) Immunoblotting for azurin levels in tumor tissues.
(E) Immunofluorescence staining of tumor sections. The azurin expressed and secreted by US-mediated VNP20009 cells was stained with an anti-FLAG antibody (red). Bacterial cells were stained with an anti-DnaK antibody (green). Nuclei were stained with DAPI (blue). Azurin-FLAG and DnaK were detected by fluorescence confocal microscopy. Scale bar, 10 μm.
See also Figures S5–S7.