FIGURES LEGENDS
Figure 1. ISX9 activates Wnt/β-catenin signaling in HEK293T cells. (A) Screening of chemical compounds that activate Wnt/β-catenin signaling. HEK293T cells carrying a Wnt responsive luciferase reporter (SuperTOPFlash) were treated with LiCl and individual small molecule compounds. The cells were lysed and luciferase activities were quantified. (B) Chemical structure of ISX9. (C) HEK293T cells were transfected with SuperTOPFlash or SuperFOPFlash reporter constructs as indicated. After transfection for 24 h, the cells were treated with vehicle control (DMSO) or increasing concentrations of ISX9 (2.5-40 µM) for another 24 h, then luciferase values were normalized to β gal activities. (D) HEK293T cells were transfected with SuperTOPFlash or SuperFOPFlash reporter constructs as indicated. After transfection for 24 h, the cells were treated with vehicle control or LiCl (20 and 40 mM) for another 24 h, then luciferase values were normalized to β gal activities. (E) HaCat cells were transfected with SuperTOPFlash or SuperFOPFlash reporter plasmids for 24 h. The cells were then treated with vehicle control or increasing concentrations of ISX9 (2.5-40 µM) for another 24 h and finally, the luciferase values were normalized to β‐gal activities. Each treatment was performed in six replicates (n = 6).
Figure 2. ISX9 increases the expression levels of active β-catenin and total β-catenin, but had little effect on expression and activity of LRP6 and GSK3β. HEK293T (A), HaCat (B), and NIH3T3 (C) cells were treated with either vehicle control (DMSO) or increasing doses of ISX9 (2.5-40 µM) for 24 h. The endogenous expression levels of active β-catenin, total β-catenin, phosphorylated LRP6, total LRP6, phosphorylated GSK3β, total GSK3β, and CK1ε, were detected by western blotting.
Figure 3 : ISX9 potentiates Axin1/LRP6 association to activate the Wnt/β-catenin pathway. (A) (Upper panel) Schematic representation of the mammalian two-hybrid system used in this study. (Lower panel). The reporter plasmid UAS-TK-Luc was transfected into HEK293T cells with a VP16-Axin1 expression plasmid, either alone or with VP16-Axin1 and Gal4-LRP6. Transfected cells were treated with different ISX9 concentrations (5 and 10 μM) as indicated. The cells were harvested at 24 h after treatment, and then luciferase activities were determined. (B) The reporter plasmid UAS-TK-Luc was transfected into HEK293T cells with a VP16-Axin1 expression plasmid, either alone or with VP16-Axin1 and Gal4-LRP6. Transfected cells were treated with different LiCl concentrations (20 and 40 mM) as indicated. The cells were harvested at 24 h after treatment, and then luciferase activities were determined. (C) Exogenous immunoblotting was performed using HEK293T cells. The cells were co-transfected with LRP6-V5 and Axin1-Flag expression plasmids for 24 h. The cells were incubated with the indicated treatment of vehicle control (DMSO) or ISX9 (5 and 10 µM) for another 24 h. The lysates were immunoprecipitated with anti-flag beads. Endogenous immunoblotting was performed using (D) HEK293T and HaCat (E) cells. Immunoprecipitation of cell lysates were performed using control IgG or anti-Axin1 antibody. Immunoblotting was carried out using the indicated antibodies. The images shown are representative of data generated in at least three independent experiments.
Figure 4 . ISX9 promotes the association of LRP6-ICD with Axin1 through covalently binding to the N-terminal region of Axin1. The binding of fluorescently labeled Axin1-300 (A), Axin301-521 (B), Axin522-862 (C), LRP6-ICD (D) to ISX9 were analyzed with a MST assay. The ISX9 is titrated from 0.61 nM to 5 μM. The change in the thermophoretic signal leads to a Kd of 595 nM. (E) The emission spectra of the ISX9 in the presence of increasing concentrations of Axin1-300 fragment (0-9 mg/mL) (λex = 405 nm). (F) MS spectra of protein-ligand complex formed between Axin1-300 and ISX9. The blue arrow indicated molecular weight of protein, red arrow indicated molecular weight of the protein-ligand complex. The molecular weight of ligand ISX9 was calculated as 233.44.
Figure 5. ISX9 upregulates the expression of Wnt target genes and stemness marker genes. HEK293T (A), HaCat (B), and NIH3T3 (C) cells were incubated with vehicle control (DMSO) or the indicated concentrations of ISX9 (2.5-40 µM) for 24 h. Total RNA was extracted and cDNAs were obtained by reverse transcription, and then subjected to real-time PCR analysis to detect the mRNA expression of Axin2, LEF1, Fibronectin and Survivin. The results were shown as mean ± SD from three independent experiments. HEK293T (D), HaCat (E), and NIH3T3 (F) cells were treated with vehicle control (DMSO) or the indicated concentrations of ISX9 (2.5-40 µM) for 24 h. Protein expression of Axin2, LEF1 and Survivin were detected by immunoblotting. Data shown were representative of three independent experiments. The relative expression levels of Axin2, LEF1, Fibronectin and Survivin, were quantified after normalization to GAPDH. *p < 0.05, compared to vehicle control. HEK293T (G), HaCat (H) and NIH3T3 (I) cells were incubated with vehicle control (DMSO) or the indicated amounts of ISX9 (2.5-40 µM) for 24 h. Total RNA was extracted and then reverse-transcribed into cDNA. Prepared cDNAs were subjected to quantitative PCR analysis to detect the mRNA expression of stemness marker genes SOX2, LGR5, Twist1, and OCT4. The protein levels of stemness marker genes SOX2, LGR5, and Twist1 were measured with immunoblotting after treatment with the indicated doses of ISX9 in HEK293T (J), HaCat (K) and NIH3T3 cells (L) respectively. Data shown were representative of three independent experiments. The relative expression levels of SOX2, LGR5, Twist1, and OCT4 were quantified after normalization to GAPDH. *p < 0.05, compared to vehicle control.
Figure 6. ISX9 promotes hair regrowth in vivo. C57BL/6J mice in the telogen phase (7 weeks old) were depilated. Vehicle (50%[v/v] ethanol, 30% water and 20% propylene glycol), 1% ISX9, or 2% minoxidil were topically applied daily to the dorsal skin for 25 days (n = 5 per group) (A) Representative photos of mice showed hair regrowth on mentioned days treated with each drug. (B) Quantitative measurements of hair coat recovery at the designated area from 0-25 days. (C) Gross analyses of weight of regrown hair in different groups treated for 25 days. (D) Haematoxylin and eosin (H&E) staining to assess the hair follicles of skins in different groups treated (vertical and transverse view) (E) Quantitative evaluation of the comparative number of hair follicles of HE staining images (n = 5) (F) Immunohistochemistry (IHC) staining for active and total β-catenin, Ki67 and LGR5 using the dorsal skin of mice treated with each drug for 25 days. Scale bars = 50 μm. Values are expressed as means ± SEM. *p < 0.05 and **p < 0.01, significantly different, ns, not significant.
Figure 7. ISX9 promotes the expression of hair regrowth-related markers in C57BL/6J mice. The vehicle control, 1% ISX9, or 2% minoxidil were topically applied daily to the dorsal skin for 25 days after depilation. (n = 5 per group). (A) The expression of the indicated genes, LEF1, Axin2, Fibronectin, and Survivin in each treated mouse was quantified by real-time PCR analysis on day 25. (B) The expression of the indicated gene, LGR5, Twist1, SOX2 and OCT4 in each treatment group was measured using real-time PCR analysis on day 25. (C) Immunoblotting analyses for phosphorylated LRP6, total LRP6, CK1ε, phosphorylated GSK3β, total GSK3β, active β-catenin, total β-catenin, LEF1, Axin2, Survivin, LGR5, SOX2, and Keratin17 on day 25. Values are expressed as means ± SEM. *p < 0.05 and **p < 0.01, significantly different; ns, not significant.