Figure 1.

The sinularin induces cell viability, DNA fragmentation, and caspases 3/9 in SK-HEP-1 cells. (A) Quantification of cell viability using the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay with sinularin at concentrations of 0, 0.5, 1, 5, 10, 25, 50, or 100 μM for 24, 48, and 72 h, with results expressed as the percentage of cell viability compared to untreated cells. (B) Determination of the IC₅₀ values of sinularin after 24, 48, and 72 h. R², representing coefficient of determination, and 95% confidence interval values were calculated. (C) Concentration-response S curves for sinularin after 24, 48, and 72 h of incubation. The cell proliferation inhibition response (y-axis), and logarithmic concentration (x-axis) in the presence of different sinularin concentrations. R² and 95% confidence interval values of each concentration curve are shown in Figure 1B. (D) Flow cytometric analyses of apoptosis using Annexin-V/PI staining in the cells treated with sinularin after 24 h. The dot-plot quadrant diagram reflects the Annexin-V (x-axis; green) and PI (y-axis; red). The lower left quadrant shows the result for normal cells (Annexin V−/PI−) and the upper left quadrant shows the result for necrotic cells (Annexin V−/PI+), while the lower right quadrant shows the result for early apoptotic cells (Annexin V+/PI−) and the upper right quadrant shows the result for late apoptotic cells (Annexin V+/PI+). (E) Quantification of the Annexin V+/PI− (early apoptotic cells) and Annexin V+/PI+ (late apoptotic cells) regions. (F) Immunofluorescence showing apoptotic bodies in the SK-HEP-1 cells (marked in green) by the TUNEL-BrdU assay after being treated with 0, 1, 5, or 10 μM of sinularin for 24 h. DAPI staining was used to observe cellular DNA/nuclei (blue) and was visualized under a fluorescence microscope (200× magnification). (G) Quantification of TUNEL-positive cells. (H) A representative Western blot showing bands of caspases 3/9 in their pro- and cleavage forms, together with the housekeeper protein β-actin after being treated with sinularin for 24 h. Full, uncropped Western blot gels are shown in Supplementary Figure S2A. (I) Pro- and cleaved caspase 9 levels quantified with densitometry analysis using ImageJ software after normalized with the β-actin level. (J) The pro- and cleaved caspase 3 levels quantified with densitometry analysis using ImageJ software after being normalized with the β-actin level. (K) After prior incubation with Z-DEVD-fmk (5 μM) for 2 h, SK-HEP-1 cells were treated without or with sinularin (10 μM) for an additional 24 h and then examined by cleaved caspase-3 kit and ELISA read analysis. Each bar represents the mean ± SE of three independent experiments. The results were analyzed using Student’s t-test and ANOVA to determine the significance. * p < 0.05 and ** p < 0.01 relative to the control (sinularin-untreated cells), and ^# p < 0.01 relative to the experimental group with 10 μM of sinularin alone.