Table 1.
Properties of the acoustic unit.
Table 2.
A summary of the experimental procedures in dual-treatment protocols.
Figure 1.
Ultrasound-induced cell killing in MES-SA and MES-SA/DX5 cells 24 hr post exposure to different acoustic intensities.
(a) Cell viability assessed by WST-8 and cell counting assay. Asterisks (*) indicate the statistical significance of the difference between the absolute percentages obtained from WST-8 and cell counting assays at one intensity for one cell line. (b) Flow cytometric analyses for FITC-labelled Annexin V and PI staining. Vertically-aligned asterisks indicate the statistical significance of Annexin V (+)/PI (−) cells, whereas horizontally-aligned asterisks indicate the statistical significance of Annexin V (+)/PI (+) cells in comparison to control. Data points are presented as mean ± SEM.
Table 3.
A summary of the differential responses of MES-SA and MES-SA/DX5 cells to US treatment.
Table 4.
The correlation coefficients (r2) between WST-8 assay and other tests employed to evaluate the extent of cell killing in uterine sarcoma cells.
Figure 2.
Proliferation of MES-SA and MES-SA/DX5 cells over 7 days (D1–D7) after sonication.
Ultrasound was applied at an intensity of 0.4 W/cm2 on D0 (arrow). Cells were counted immediately after sonication and plated at a density of 1×105 cells/dish. Data points are presented as mean ± SEM.
Figure 3.
Pictograms of MES-SA (a & b) and MES-SA/DX5 (c & d) cells 24 hr post sonication at 0.4 W/cm2. Cells were stained simultaneously with Hoechst 33342 and Alexa flour-488 – conjugated wheat germ agglutinin (WGA) in 2% paraformaldehyde (PFA)/PBS for 20 min followed by immediate observation. Cells show nuclear budding of genomic content (d) that is occasionally translocated through the cytoplasm (b) and emerges to form a small cell (Raju cell) (a). Two distinct forms of nuclear budding can be identified in treated MES-SA cells bearing similarity to neotic cytokinesis described by Rajaraman et al. [33], namely, (a) sequential cytokinesis type-2 perinuclear and (b) sequential cytokinesis type-1 pericellular, whereas the first form only was observed in treated MES-SA/DX5 cells. Open-head arrows indicate nuclear budding; closed-head arrows indicate the surrounding membranes of emerging cells. (e & f) Sonicated MES-SA cells showing multiple cytokinesis captured at the fourth (D4) and fifth (D5) days post exposure, respectively. (g) Control MES-SA cell with spontaneous cytokinesis. Cells (e–g) resemble in morphology neotic mother cells shown by Sundaram et al. [31]. Bars, 10 µm.
Figure 4.
The extent of histone H2AX phosphorylation in MES-SA and MES-SA/DX5.
Cells were fixed 15 min after exposure to ultrasound at different intensities. Cells were assayed flow cytometrically. Data points are presented as mean ± SEM. Asterisks (*) denote the statistical significance between MES-SA and MES-SA/DX5 at respective intensities.
Figure 5.
Cell cycle analysis of MES-SA and MES-SA/DX5 cells.
(a) The fraction of polyploid cells detected 1 hr and 24 hr after sonication at 0.4 W/cm2. Data points are presented as mean ± SEM. Asterisks (*) denote statistical significance. (b) Flow cytometric dot plots of forward (y-axis; FS) versus side (x-axis; SS) scatter parameters showing the increase in cell size of MES-SA/DX5 cells 1 hr post sonication at 0.4 W/cm2. (c) Cell cycle analysis performed 24 hr post sonication at different intensities.
Figure 6.
Cell survival following dual treatment protocols with doxorubicin (1 µM) and ultrasound (0.4 W/cm2). (a) Simultaneous treatment protocols. (b) Sequential treatment protocols. Data points are presented as mean ± SEM. Asterisks (*) denote the statistical significance of changes in cell survival following (Dox-US) protocol compared to (US 48) and (Dox 48) for each cell line. Following (US-Dox) treatment protocol, the cell survival of MES-SA cells decreased significantly compared to (US 48) but was insignificantly increased compared to (Dox 48). In MES-SA/DX5 cells, cell survival was unchanged. However, (US-Dox) protocol showed consistently large difference between the percentages obtained from WST-8 and cell counting assays indicating the presence of cells contributing to viability below the size threshold for detection by the cell counter.