Fig 2: NCTD suppresses NRF2/HO-1 signaling in ovarian cancer cells. SKOV3 and OVCAR-3 cells were treated with 10 and 20 µM NCTD for 24 h. Reverse transcription-quantitative PCR analysis indicated that the mRNA levels of NRF2, HO-1, xCT and GPX4 were significantly suppressed in the (A) SKOV3 and (B) OVCAR-3 cells treated with NCTD. (C) Western blot analysis demonstrated that the NRF2, HO-1, xCT and GPX4 protein levels were significantly suppressed in SKOV3 and OVCAR-3 cells incubated with NCTD. An unpaired Student's t-test was used for statistical analysis. *P<0.05, **P<0.01 and ***P<0.001 vs. control (DMSO). NCTD, norcantharidin; NRF2, nuclear factor erythroid 2-related 2; HO-1, heme oxygenase 1; xCT, solute carrier family 7 member 11; GPX4, glutathione peroxidase 4.

Fig 3: HCA supplementation markedly suppressed ferroptosis in HUVEC cells after H/R stimulation. (A) Immunoblots for ASCL4, SLC7A11, GPX4, and FTH1 in the indicated group treated either with erastin (4 μM) or Fer-1 (1 μM). (C) Western blots for HIF-1α, HO1, SLC7A11, and GPX4 protein of HUVEC cells in the indicated group. (B,D) Quantification of the results of (A,C). (E) Relative mRNA expression of HIF-1α, HO-1, SLC7A11, and GPX4 in the indicated group. n = 5 per group. (Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with indicated group).

Fig 4: Eng inhibited RANKL-induced ROS activation and increased the expression of ROS scavenging enzymes. (A) RAW264.7 cells were induced with RANKL (50 ng/mL) and different concentrations of Eng (10, 20,40 μM) for 48 h, and then incubated with 5 μM DCFH-DA in the dark for 30 min. The fluorescence of DCF was observed under a laser confocal microscope. Scale bar = 300μm. (B) Quantification of DCF fluorescence intensity in an average per cell. (C) RAW264.7 were stimulated with RANKL (50 ng/mL) and different concentrations of Eng for 48 h. Representative Western blotting images of the effects of Eng on Nrf2, HO-1 and NQO-1 expression. (D) Quantification of the ratios of band intensity of Nrf2, HO-1, and NQO-1 relative to β-actin. All bar charts are presented as mean ± SD; n=3. *P<0.05, **P<0.01, ***P<0.001, ns: not statistically significant, vs RANKL group.

Fig 5: Inhibitory effect of PtAu2 clusters on LPS-induced calvarial osteolysis in vivo. (A) Schematic diagram of in vivo experiments. (B) Representative micro-CT scanning images of mice calvaria. (C) Quantitative analysis of micro-CT scanning images. (D) Representative H&E, TRAP, and immunostaining images of TNF-α, Nrf2, and HO-1. (E) Quantification of the number of TRAP-positive OCs. (F) Quantification of OcS/BS%. (G) Quantitative analysis of relative TNF-α fluorescence. (H) Quantitative analysis of relative Nrf2 fluorescence. (I) Quantitative analysis of relative HO-1 fluorescence. (C) Data represent the mean ± SD of five independent animals (one-way ANOVA with Tukey's post hoc test). (E, F, G, H, and I) Data represent the mean ± SD of four independent animals (one-way ANOVA with Tukey's post hoc test). *p < 0.05; **p < 0.01.