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. 2021 Nov 12;12:789552. doi: 10.3389/fphar.2021.789552

FIGURE 5.

FIGURE 5

Nontoxic PNC suppressed human breast cancer MBA-MD-231 cell-induced osteoclastogenesis, promoted apoptosis of MBA-MD-231 cells, and inhibited the migration as well as invasion of MBA-MD-231 cells in vitro. (A) Viability of MBA-MD-231 cells after PNC treatments for 24–96 h (n = 3). (B) Flow cytometric analysis of apoptotic breast cancer cells with PNC treatment. (C) Quantification of the percentage of apoptotic MDA-MB-231 cells after treatment with diverse concentrations of PNC (0, 62.5, 125,250 μM) for 48 h (n = 3). (D) RAW 264.7 cells cocultured with MDA-MB-231 cells, treated with PNC, and cocultured for 1 week before TRAP staining. (E) Quantitative analysis of TRAP positive osteoclasts (n = 3). (F) Efficacy of PNC on the suppression of migration as well as invasion of MDA-MB-231 cells. The irregularly shaped cells, that invaded and migrated, showing purple staining were counted and photographed. (G) Quantification of the percentage of cell invasion and migration by ImageJ software. Bars represent the SD, whereas columns represent the means (n = 3). (H) After the PNC-treated MDA-MB-231 cells were cultured for 2 days, PNC-induced cleaved caspase-3 and Bcl-2 levels were examined using Western blotting. (I) Average ratios of cleaved caspase-3 as well as Bcl-2 relative to β-actin. **p < 0.05, **p < 0.01, when compared to the control group.