Figure 3.
Cytotoxic effects of azurin secreted by US-mediated VNP20009 cells against melanoma and colorectal cancer cells
(A) Schematic representation of SINGER-mediated protein expression and secretion in VNP20009 cells.
(B) The expression and secretion of a Gaussia luciferase (Gluc) reporter in VNP20009 cells after US stimulation.
(C) Schematic for the time schedule and experimental procedure for assessing the reversibility of the SINGER system.
(D) Reversibility of SINGER-mediated transgene expression. VNP20009 cells (OD600 = 0.25) transformed with pGT240 were cultivated for 36 h while alternating irradiation with US (0.5 W/cm2, pulse of 1 s on, 1 s off) for 1 h or without US. Gluc expression in the culture supernatants was quantified every 2 h. Cell density was adjusted to OD600 = 0.25 when the pattern changed.
(E) Schematic of the experimental procedure for analysis of secretion proteins.
(F and G) The expression and secretion of azurin in VNP20009 cells after US stimulation. Azurin in the culture supernatants was checked by immunoblotting analysis (F) and ELISA (G).
(H) Measurement of cell viability by CCK-8 assays. B16F10 and CT26 cells were treated with the culture supernatants of VNP20009 harboring SINGER-azurin with (right) or without (left) US stimulation.
(I) Fluorescence microscopy images for the detection of apoptosis by TUNEL assay. Scale bar, 50 μm.
(J–L) Representative flow cytometry plots using annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining for apoptosis (J and K) and quantification of apoptosis (L).
Data in (B), (D), (G), (H), and (L) are presented as means ± SD; n = 3 independent experiments. Each data point represents the mean of three technical replicates. p values were calculated by one-way analysis of variance (ANOVA) with Tukey’s post-test. ∗∗∗p < 0.001.
See also Figures S2–S4.