Article Figures & Data
Figures
- Fig. 1.
The electrostatic potentials of receptor and ligands. (A) The electrostatic potentials of AG490 and the pocket of JAK2. (B) The electrostatic potentials of dehydrocrenatidine and the pocket of JAK2. Dehydrocrenatidine can insert into the deeper position of JAK2 domain according to an analysis of molecular shape and electrostatic potential.
- Fig. 2.
Dehydrocrenatidine inhibited STAT3-hyperactivated cancer cell survival and STAT3 phosphorylation without affecting P65 and Akt phosphorylation. hTERT-BJ, DU145, MDA-MB-468, and MCF 10A cells were treated with (A) dehydrocrenatidine, (B) AG490, (C) AZD1480, and (D) staurosporine at indicated concentrations for 48 hours, and cell viability was measured by MTT assay. (E) DU145 and MDA-MB-468 cells were treated with DMSO, dehydrocrenatidine (10 μM), Ly294002 (50 mM), and TPCA-1 (1 μM) for 2 hours and Western blotted.
- Fig. 3.
Dehydrocrenatidine inhibited STAT3 phosphorylation in DU145 and MDA-MB-468 cells in a dose- and time-dependent manner. (A) DU145 and MDA-MB-468 cells were treated with indicated concentrations of dehydrocrenatidine for 2 hours, and cell lysates were blotted by indicated antibodies. DMSO was used as control. p-STAT3/STAT3 relative density was measured by Image J, and IC50 was calculated by SPSS software. (B) DU145 and MDA-MB-468 cells were treated with 10 μM dehydrocrenatidine for indicated time points, and cell extracts were analyzed by Western blot. DMSO was used as control. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
- Fig. 4.
Dehydrocrenatidine inhibited IL-6 and IFN-induced STAT3 phosphorylation and their downstream gene expression. (A) Hela, HepG2, and HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium with 0.2% FBS for 12 hours. Serum-starved cells were pretreated with DMSO or dehydrocrenatidine (10 μM) for 1 hour and stimulated by IL-6 (250 ng/ml) for 2 hours, and cell lysates were blotted by indicated antibodies. (B) Serum-starved Hela cells were incubated with DMSO or dehydrocrenatidine (10 μM) for 1 hour and treated with IFNγ (150 IU/ml) or (C) IFNα (5000 IU/ml) for 2 hours. Cells lysates were blotted by indicated antibodies. (D) Serum-starved Hela cells were incubated with DMSO or dehydrocrenatidine with indicated concentrations for 1 hour and stimulated with IFNα (5000 IU/ml) for 2 hours. Cell lysates were blotted by indicated antibodies. p-STAT1/STAT1 relative density was measured by Image J, and IC50 was calculated by SPSS software. (E) Hela and (F) HepG2 cells were serum-starved and pretreated with DMSO or dehydrocrenatidine (10 μM) for 1 hour and stimulated by IL-6 (250 ng/ml) for 4 hours. mRNA levels of socs3 were analyzed by real-time PCR. (G) Serum-starved Hela cells were pretreated with DMSO or dehydrocrenatidine (10 μM) for 1 hour and stimulated by IFNα (5000 IU/ml) for 4 hours, and irf1 mRNA levels were analyzed by real-time PCR. Data are mean ± S.D. of three independent experiments. ***P < 0.001, one-way analysis of variance. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
- Fig. 5.
Dehydrocrenatidine inhibited overexpression of the JH1 domain of JAK1-, JAK2-, and TYK2-induced protein phosphorylation. (A) HEK293T cells transfected with JH1 domain of JAK1, JAK2, and TYK2 were treated with dehydrocrenatidine (15 μM) for 4 hours and blotted by indicated antibodies. (B) Serum-starved Hela cells were pretreated with DMSO, dehydrocrenatidine (15 μM), or lapatinib (10 μM) for 1 hour and stimulated with EGF (50 ng/ml) for 30 minutes, and cell lysates were Western blotted. (C) HEK293T cells overexpressing c-Src were incubated with dehydrocrenatidine (15 μM) or dasatinib (500 nM) for 4 hours. Cell lysates were analyzed by Western blot. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
- Fig. 6.
Dehydrocrenatidine induced apoptosis. (A) DU145 and (B) MDA-MB-468 cells were plated on coverslips. After 12 hours, medium was replaced and DMSO (a and c) or dehydrocrenatidine (10 μM) (b and d) was added. After 16 hours of treatment, cells were stained with Hoechst and visualized under a microscope (a and b, 40× magnification; c and d, 20× magnification). (C) DU145 and MDA-MB-468 cells were treated with DMSO or dehydrocrenatidine (10 μM) for 24 hours, double stained by annexin V and propidium iodide, and flow cytometry was performed. (D) DU145 and MDA-MB-468 cells were treated with dehydrocrenatidine (10 μM) for 24 or 48 hours. Cell lysates were analyzed by Western blot. DMSO was used as control. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
- Fig. 7.
Dehydrocrenatidine inhibited JAK2-JH1–induced STAT3 phosphorylation. (A) Recombined hSTAT3 protein and purified JAK2-JH1 kinase domain protein were used for kinase assay. Dehydrocrenatidine was applied at the following concentration series: 20, 40, 80, 100, or 200 μM. p-STAT3 levels were analyzed by Western blot. (B) Three independent kinase assays were performed. p-STAT3/STAT3 relative densities were quantified by Image J. Data represent mean ± S.D. of three independent experiments.
Additional Files
Data Supplement
Files in this Data Supplement:
- Supplemental Data -
Supplemental Figure 1 - The structures of ranking top 5 molecules
Supplemental Figure 2 - Western blot confirmation of activity of the top ranking molecules
Supplemental Figure 3 - 1H-NMR spectrum for Dehydrocrenatidine
Supplemental Figure 4 - MS for Dehydrocrenatidine
Supplemental Figure 5 - HPLC for Dehydrocrenatidine
Supplemental Figure 6 - Docking results between JAK1, JAK2, JAK3, TYK2 and Dehydrocrenatidine
Supplemental Figure 7 - K562 cells were cultured in RMPI-1640 with 0.2% FBS for 12 hours...
Supplemental Table 1 - The structures of JAK2 inhibitors
Supplemental Method 1
References
- Supplemental Data -
In this issue
Dehydrocrenatidine Is a Novel JAK Inhibitor
