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. 2020 Oct 12;11:5127. doi: 10.1038/s41467-020-18929-0

Fig. 3. VAL is important for activated AKT-induced LAD metastasis.

Fig. 3

a, b Representative images and quantification of invading cells in five random fields of Matrigel-coated transwell assay. c Relative luciferase activities of myr-AKT1-overexpressing LAD cells silenced with VAL or corresponding vector-control cells adhering to the Matrigel. d The sub-G1 DNA contents of detached cells for myr-AKT1-overexpressing LAD cells silenced with VAL or corresponding vector-control cells are shown in the anoikis assay. e H&E staining of both the dermal tissue and tumor tissue in the subcutaneous tumors xenografted with the indicated cells. Black arrows mark the direction of tumor invasion with invasive front marked by dashed line. f Myr-AKT1-overexpressing LAD cells silenced with VAL or corresponding vector-control cells labeled with luciferase expression were injected via cardiac ventricle into nude mice (n = 5 per group). Representative bioluminescent images of systemic metastasis are shown. g Frequencies of formation of metastatic lesions in nude mice (n = 5 per group) intracardially injected with different cell numbers of the indicated cells. h Metastatic bone lesions in mice intracardially injected with the indicated cells were confirmed by micro-CT imaging and H&E staining. i Myr-AKT1-overexpressing LAD cells were intracardially injected into nude mice (n = 5 per group), followed by intravenous injection with chemosynthetic VAL siRNAs or control scramble siRNAs. Representative bioluminescence images of systemic metastasis are shown, and quantitation of bioluminescent intensities as analyzed by ROI tools. j The effect of intravenous injection of VAL siRNAs on the formation of metastatic bone lesions in nude mice intracardially injected with A549-myr-AKT1 cells was evaluated by micro-CT imaging and H&E staining. k Kaplan–Meier analysis (Log-rank test) of the effect of antagonizing VAL on the metastasis-free survival and overall survival of nude mice intracardially injected with myr-AKT1-overexpressing LAD cells. Scale bar: 100 μm (a, e, h, j). Scatter dot plots represent the means ± SD derived from three independent experiments. Statistical analyses were performed by two-way ANOVA multiple comparison analysis (b–d) and two-tailed unpaired Student’s t test (i). Source data are provided as a Source Data file.