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Figure A1. The stability of PHNQ extract in cell culture media. The percentage of the original absorbance at 340 nm of PHNQ extract (in cell culture media at the concentration of 62.5 µg/mL) at different time points in media at room temperature either in the dark or under the light. Means with different letters for the group under light (a–f) and in the dark (A–F) are significantly different (p < 0.05).
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Figure 1. Structure of major polyhydroxylated naphthoquinones (PHNQs) in E. chloroticus spine. (a) spinochrome E; (b) spinochrome B; (c) echinochrome A.
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Figure 2. Absorbance data measured at 570 nm of Saos-2 cells treated with different concentrations of PHNQ extract as determined using the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay as described in Section 4.4 after 48 h incubation. Data represent the mean ± standard deviation of three independent experiments, each concentration tested in duplicates. Means with different letters for the group with different concentrations are significantly different (p < 0.05). DMSO (3.725% v/v) was used as vehicle control. Sodium dodecyl sulfate (SDS) at 10% (w/v) in final media was used as a positive control, for which no viable cells were observed after 48 h incubation.
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Figure 3. Absorbance data measured at 570 nm of Saos-2 cells treated with different concentrations of CaCl2 (A), echinochrome A and PHNQ (B), as determined using the MTT assay as described in Section 4.5 after 21 days of incubation. Data represent the mean ± standard deviation of three independent experiments; each concentration was tested in duplicates. Means with different letters are significantly different (p < 0.05), determined by one-way ANOVA. Sodium dodecyl sulfate (SDS) at 10% (w/v) in final media was used as a positive control (no viable cells were evident). EchA refers to echinochrome A, with H: 62.5 µg/mL; M: 31.25 µg/mL; L: 15.625 µg/mL. PHNQH, PHNQM and PHNQL refer to PHNQ at concentrations of 62.5, 31.25 and 15.625 µg/mL, respectively.
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Figure 4. Absorbance data measured at 570 nm of Saos-2 cells treated with echinochrome A (A) and PHNQ extract (B) with different concentrations of CaCl2, as determined using the MTT assay as described in Section 4.4 after 21 days incubation. Data represent the mean ± standard deviation of three independent experiments; each concentration was tested in duplicates. Columns with different letters are significantly different (p < 0.05) for different concentrations of echinochrome A or PHNQ, determined by one-way ANOVA. Sodium dodecyl sulfate (SDS) at 10% (w/v) in final media was used as a positive control, with no viable cells evident. Cytotoxicity of PHNQ on Saos-2 cells was measured by MTT assay. Same volume of DMSO was used as treatment group for the treatment in vehicle control group.
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Figure 5. Formation of mineralized nodules by Saos-2 cells treated with different concentrations of CaCl2. (A) measured as a percentage of control (with excitation and emission wavelengths of 440 and 610 nm, respectively), after staining with xylenol orange (at 20 μmol for 24 h) at different timepoints. Percentage of control was calculated according to the quotation in Section 4.5. (B) Measured as a percentage of control after staining with xylenol orange (at 20 μmol for 24 h) at different timepoints (Quantitative image analysis by ImageJ, see details in Appendix A). Percentage of control was calculated according to the quotation in Appendix A. (C) Measured as a percentage of control after staining with von Kossa (Quantitative image analysis by ImageJ). Percentage of control was calculated according to the quotation in Appedix A. Data represent the mean ± standard deviation of three independent experiments, each concentration was tested in duplicate. Means with different letters are significantly different (p < 0.05), determined by one-way ANOVA using Tukey’s honesty test at p-value < 0.05. Cells only (CaCl2 = 0 mM) were used as a control.
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Figure 6. Representative examples of stained images of the formation of mineralized nodules by Saos-2 cells treated with CaCl2 at 1, 2, 3, 4 mM. Cells stained with either (A) xylenol orange (at 20 μmol for 24 h) at day 20 or using the (B) von Kossa method (silver nitrate) at day 21. Images were taken with a Nikon DS-Qi2 Camera fitted to a Nikon (ECLIPSE, Ti2) inverted fluorescent (or light) microscope. An exposure time of 166 ms was used for xylenol orange.
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Figure 7. Formation of mineralized nodules by Saos-2 cells treated with different concentrations of (A) Echinochrome A and (B) PHNQ extract. Measured as a percentage of control (with excitation and emission wavelengths of 440 and 610 nm, respectively), after staining with xylenol orange (at 20 μmol for 24 h). Data represent the mean ± standard deviation of three independent experiments, each concentration was tested in duplicate. Means with different letters on the same timepoint are significantly different (p < 0.05), as determined one-way ANOVA using Tukey’s honesty test at p-value < 0.05. Cells-only were used as a control (EchA: echinochrome A). Percentage of control was calculated according to the quotation in Section 4.6.
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Figure 8. Representative stained images of the formation of mineralized nodules by Saos-2 cells treated with different concentrations of PHNQ extract and cells-only control. Stained with either (A) xylenol orange (at 20 μmol for 24 h) at day 20 or using (B) the von Kossa method (silver nitrate) at day 21. Images were taken with a Nikon DS-Qi2 Camera fitted to a Nikon (ECLIPSE, Ti2) inverted fluorescent (or light) microscope. An exposure time of 166 ms was used for xylenol orange. (PHNQH: 62.5 µg/mL PHNQ; PHNQM: 31.25 µg/mL PHNQ; PHNQL: 15.625 µg/mL PHNQ).
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Figure 9. Formation of mineralized nodules by Saos-2 cells treated with PHNQH and different concentrations of CaCl2. (A) PHNQH + 1.5 mM CaCl2 (B) PHNQH + 2.0 mM CaCl2 (C) PHNQH + 2.5 mM CaCl2 (compared with PHNQ extract alone and CaCl2 alone. Measured as a percentage of the control (with excitation and emission wavelengths of 440 and 610 nm, respectively), after staining with xylenol orange (at 20 μmol for 24 h). Data represent the mean ± standard deviation of three independent experiments, each concentration was tested in duplicate. Means with different letters are significantly different (p < 0.05), as determined by one-way ANOVA using Tukey’s honesty test at p-value < 0.05.
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Figure 10. Representative stained images of the formation of mineralized nodules by Saos-2 cells treated with PHNQH, CaCl2 and PHNQH + CaCl2. Stained with either (A) xylenol orange (at 20 μmol for 24 h) at day 20 or using (B) the von Kossa method (silver nitrate) at day 21. Images were taken with a Nikon DS-Qi2 Camera fitted to a Nikon (ECLIPSE, Ti2) inverted fluorescent (or light) microscope. An exposure time of 166 ms was used for xylenol orange. (PHNQH: 62.5 µg/mL PHNQ).
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Figure 11. Formation of mineralized nodules by Saos-2 cells treated with PHNQM and PHNQL with added different concentrations of CaCl2. (A) PHNQM + 1.5 mM CaCl2 (B) PHNQM + 2.0 mM CaCl2 (C) PHNQM + 2.5 mM CaCl2 (D) PHNQL + 1.5 mM CaCl2 (E) PHNQL + 2.0 mM CaCl2 (F) PHNQL + 2.5 mM CaCl2 compared with PHNQ extract alone and CaCl2 alone. Measured as percentage of control (with excitation and emission wavelengths of 440 and 610 nm, respectively), after staining with xylenol orange (at 20 μmol for 24 h). Data represent the mean ± standard deviation of three independent experiments, each concentration was tested in duplicate. Means with different letters are significantly different (p < 0.05), as determined by one-way ANOVA using Tukey’s honesty test at p-value < 0.05. Cells only (CaCl2 = 0 mM) at day 10 used as a control.
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