SS induces breast cancer cell apoptosis, G2/M checkpoint arrest, ROS accumulation and LDH-A inhibition.

(A) Annexin-Cy5 staining results showed that SS induced both breast cancer cells apoptosis in a dose-dependent manner; (B) JC-1 staining assay indicated that mitochondrial membrane potential was decreased after SS treatment, indicating that the mitochondrial pathway apoptosis was triggered by SS; (C) Cell cycle analysis showed that after SS treatment, the G2/M checkpoint was arrested in both breast cancer cell lines, presenting as a significant increase in G2/M subpopulations; (D) Hydroethidine was applied to detect the intracellular O₂⁻ level after SS administration by flow cytometry. The results showed that the intracellular O₂⁻ level was increased after SS treatment for 24 h (upper panel). Clark oxygen electrode was applied to detect the oxygen consumption speed of breast cancer cells after SS treatment. The results showed that the oxygen consumption speed of both breast cancer cells was rapidly enhanced after SS administration (lower panel); (E) LDH-A activity assay showed that SS were dose- and time- dependent when suppressing the LDH-A activity; (F) Western blotting results indicated that after SS administration, the expression of LDH-A in both breast cancer cells under both normoxia and hypoxia condition were down-regulated.