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. 2025 Jul 28;21(11):4872–4894. doi: 10.7150/ijbs.113785

Figure 2.

Figure 2

Fe(hino)3 inhibited osteosarcoma cell proliferation and promoted cell death. (A) Cell viability of osteosarcoma cells treated with different concentrations of Fe(hino)3 (0, 0.5, 1.0, 2.0, 4.0, 6.0 μM) for 24 h, as measured by CCK-8 assay. (B) Cell viability of osteosarcoma cells treated with different concentrations of hinokitiol (0, 5, 10, 20, 30, 40, 50, 60 μM) for 24 h. (C) Prussian blue staining showing intracellular iron accumulation after treatment with FeCl3 (1.6 μM for 143b, 1 μM for HOS), hinokitiol (4.8 μM for 143b, 3 μM for HOS), or Fe(hino)3 (1.6 μM for 143b, 1 μM for HOS) for 24 h. Scale bar = 100 μm. (D) Morphological changes of osteosarcoma cells after treatment with indicated compounds for 24 h. Scale bar = 100 μm. (E) Colony formation assay showing the long-term effects of Fe(hino)3 on osteosarcoma cell proliferation. (F) Flow cytometry analysis of cell apoptosis using Annexin V/PI staining after Fe(hino)3 treatment for 24 h. (G-I) Western blot analysis and quantification of apoptosis-related proteins after Fe(hino)3 treatment (1, 1.6, 3 μM for 143b; 0.75, 1, 2 μM for HOS) for 24 h. (J, K) Cell viability after treatment with Fe(hino)3 in combination with different cell death inhibitors. Cells were pretreated with DFO (50 μM, 6 h), NAC (5 mM, 1 h), Fer-1 (1 μM), z-VAD-FMK (25 μM, 1 h), or VX765 (30 μM, 1 h) before Fe(hino)3 treatment. Data are presented as mean ± SD from three independent experiments. *P < 0.05, **P < 0.01 vs control group.