Sulfuretin stimulates chondrocyte differentiation in cell culture. (A) C3H10T1/2 cells were differentiated into chondrocytes in the presence of sulfuretin (10 or 20 μM) for 12 days followed by Alcian Blue staining. (B) Relative mRNA expression levels of early chondrocyte genes (Col2a1, Aggrecan, and Sox9), late chondrocyte genes (Col10a1, Mmp3, and Mmp13), and adipocyte genes (Pparg, Fabp4, and Cd36) after sulfuretin treatment were determined by real-time PCR. (C) Primary chondrocytes were isolated from mice at postnatal day 7 and treated with sulfuretin for 12 days. Relative mRNA expression was quantified by real-time PCR. Data shown represent mean ± SEM. Statistical significance was determined relative to a control by Student’s t-test (*P < 0.05; **P < 0.01).

Sulfuretin stimulates Nrf2 activity. (A) C3H10T1/2 cells were treated with sulfuretin (20 μM) for 0.5, 1, 2, 6, 12, and 24 hours and relative temporal expression levels of Hmox1 and Nqo1 were measured by real-time PCR. (B) Sulfuretin treatment at 5-40 μM for 6 hours in C3H10T1/2 cells increased Nrf2 protein levels as determined by western blotting. Actin was used for loading control and tBHQ (50 μM) was used as a control. Representative blot images and relative densitometry bar graph were shown (n = 3). (C) HEK293T cells were transfected with the Nrf2 dependent-antioxidant response element (ARE) luciferase reporter construct (6XARE) and an expression vector coding for Nrf2 (pcDNA3-Nrf2). After 24 hours, cells were treated with DMSO (control) or sulfuretin (20 μM) for 24 hours and luciferase activity was measured. Luciferase signals were normalized with Renilla luciferase activity. (D) Nrf2 wild type (WT) MEF or Nrf2 knockout (KO) MEF were treated with sulfuretin (20 μM) for 12 hours and expression levels of Nrf2 target genes were measured by real-time PCR. (E) C3H10T1/2 cells were differentiated into chondrocytes in the presence of tBHQ (100 μM) and relative levels of hypertrophic genes and Nrf2 target genes were determined by real-time PCR. Data are presented as mean ± SEM (n = 3). Statistical significance was determined by comparison to the control using Student’s t-test (*P < 0.05; **P < 0.01).

Sulfuretin treatment increases body length and Nrf2 target gene expression in zebrafish. (A) Zebrafish at 1 dpf were treated with sulfuretin (50 or 100 μM) for 4 days. Body lengths were then measured at 5 dpf. (B) Zebrafish at 1 dpf were treated with sulfuretin for 6 days and body lengths were measured at 7 dpf. (C) Total RNA was isolated from zebrafish after treatment with sulfuretin (100 μM) for 4 days and relative expression levels of mmp13a, col10a1a, col10a1b, ctgfa, and ctgfb were determined by real-time PCR. (D, E) Sulfuretin (100 μM) treatments increases the length of the ceratohyal (arrow) but not the parasphenoid (*) in zebrafish. (D) Whole mount in situ hybridization of col10a1 and ctgf. (E) Quantification of WISH staining in center of ceratohyal (col10a1), parasphenoid area (col10a1), and total length of ceratohyal (ctgf). (F) Zebrafish at 1 dpf were treated with sulfuretin for 4 days and expression levels of gclm, nqo1, gclc, and maff were measured by real-time PCR. Data are presented as mean ± SEM (n = 10 per group). Statistical significance was determined by Student’s t-test (ns, not significant; *P < 0.05; **P < 0.01).

Nrf2 is necessary for effects of sulfuretin on chondrocyte and bone growth in zebrafish. (A) Induction of chondrocyte genes by sulfuretin is blunted in Nrf2 knockdown cells. C3H10T1/2 cells were transfected with siRNA targeting Nrf2 (Nrf2 siRNA) and differentiated into chondrocytes for 12 days. Expression levels of Nrf2 targets (Hmox1 and Nqo1) and chondrocyte genes (Mmp3 and Mmp13) were measured by real-time PCR. Data are presented as mean ± SEM (n = 3). (B) Effects of sulfuretin and DMF on body lengths were compromised in nrf2 morpholino injected zebrafish. Sulfuretin (100 μM) or DMF (4 μM) was used to treat control morpholino or nrf2 morpholino injected zebrafish at 1 dpf for 4 days and their lengths were determined. Data are presented as mean ± SEM (n = 10). Statistical significance was determined by comparison with the control using Student’s t-test (ns, not significant; *P < 0.05; **P < 0.01).