Journal: Cell Death & Disease

Article Title: Nintedanib decreases muscle fibrosis and improves muscle function in a murine model of dystrophinopathy

doi: 10.1038/s41419-018-0792-6

Figure Lengend Snippet: Fibroblasts proliferation was analyzed after 2, 4, and 6 days of culture ( a–d ). We cultured fibroblast with increasing concentrations of nintedanib in a culture containing 10% fetal bovine serum (FBS) and we observed decreased fibroblast proliferation after 4 and 6 days in a dose-dependent manner ( a ). Fibroblasts were cultured in the presence of platelet-derived growth factor AA (PDGF-AA, 10 ng/mL) ( b ), basic fibroblast growth factor (FGFb, 10 ng/mL),( c) or vascular endothelial growth factor A (VEGF-A, 50 ng/mL) ( d ) with 1% FBS with or without nintedanib 0.4 μM. Only PDGF-AA increased cell proliferation, an effect that was reversed with the addition of nintedanib to the culture. Fibroblast migration was analyzed using a scratch assay. Nintedanid treatment at 0.4 μM reverted the promigratory effect of PDGF-AA, FGFb, VEGF, and CTGF ( e ). Representative images of this assay are shown in ( f ). Nintedanib treatment at 0.4 μM (blue bars) reverted the effect of PDGF-AA in the expression of ADAM metallopeptidase domain 17 ( ADAM-17) , tissue inhibitor of metalloproteinase 1 (TIMP-1) and tissue inhibitor of metalloproteinase 2 (TIMP-2) ( g ). Nintedanib treatment at 0.4 μM (blue bars) produced a statistically significant reduction of collagen type I alpha 1 chain (COL1A1) , collagen type III alpha 1 chain (COL3A1) , fibronectin 1 ( FN1) , platelet-derived growth factor A (PDGFA) , connective tissue growth factor (CTGF) , transforming growth factor beta 1 (TGFβ1) expression compared with control samples (black bars) analyzed by qPCR ( h ). Data are expressed as means ± SD. N = 3 per group. * P < 0.05, ** P < 0.01, *** P < 0.005

Article Snippet: All mRNA-specific FAM-labeled primers/probe were purchased from Applied Biosystems and detected cDNA from the following genes: Ctgf (Mm01192933_g1), Pdgfa (Mm01205760_m1), Pdgfb (Mm00440677_m1), Tgfb1 (Mm01178820_m1), Col1a1 (Mm00801666_g1), Col3a1 (Mm01254476_m1), Fn1 (Mm01256744_m1), Adgre1 (Mm00802529_m1), PDGFA (Hs00964426_m1), CTGF (Hs01026927_g1), PDGFB (Hs00966522_m1), TGFB1 (Hs00998133_m1), COL1A1 (Hs00164004_m1), COL3A1 (Hs00943809_m1), FN1 (Hs01549976_m1), ACTA (Hs00426835_g1), MMP9 (Hs00957562_m1), MMP2 (Hs01548727_m1), MMP1 (Hs00899658_m1), TIMP-1 (Hs99999139_m1), TIMP-2 (Hs00234278_m1), ADAM-12 (Hs01106101_m1), ADAM-17 (Hs01041915_m1), TP53 (Hs01034249_m1).

Techniques: Cell Culture, Derivative Assay, Migration, Wound Healing Assay, Expressing, Produced

Journal: Cell Death & Disease

Article Title: Nintedanib decreases muscle fibrosis and improves muscle function in a murine model of dystrophinopathy

doi: 10.1038/s41419-018-0792-6

Figure Lengend Snippet: Collagen type I alpha 1 chain (Col1a1), Collagen type III alpha 1 chain ( Col3a1 ), Fibronectin 1 ( Fn1 ), Platelet-derived growth factor A ( Pdgfa ), Platelet-derived growth factor B ( Pdgfb ), Connective tissue growth factor ( Ctgf ), Transforming growth factor beta 1 ( Tgfβ1 ), and Adhesion G protein-coupled receptor E1 ( Adgre1 ) showed changes in relative abundance following nintedanib in quadriceps ( a–h ), diaphragm ( i–p ), and tibialis anterior ( q–y ). Col1a1, Col3a1, Pdgfa, Tgfb1, and Adgre1 gene expression was increased in mdx mice compared with WT . Col1a1, Col3a1, Pdgfa, Pdgfb, Tgfb1, and Adgre1 expression was reduced in all muscles analyzed from nintedanib-treated mdx mice compared with mdx mice. In contrast, Ctgf expression was increased in muscles from nintedanib-treated mdx mice compared with mdx mice. Data are expressed as means ± SD. Genetic background mouse strain C57BL (WT); n = 5, mdx mice (mdx), n = 5; nintedanib-treated mdx mice (mdx + Ninte), n = 7. * P < 0.05, ** P < 0.01, *** P < 0.005

Article Snippet: All mRNA-specific FAM-labeled primers/probe were purchased from Applied Biosystems and detected cDNA from the following genes: Ctgf (Mm01192933_g1), Pdgfa (Mm01205760_m1), Pdgfb (Mm00440677_m1), Tgfb1 (Mm01178820_m1), Col1a1 (Mm00801666_g1), Col3a1 (Mm01254476_m1), Fn1 (Mm01256744_m1), Adgre1 (Mm00802529_m1), PDGFA (Hs00964426_m1), CTGF (Hs01026927_g1), PDGFB (Hs00966522_m1), TGFB1 (Hs00998133_m1), COL1A1 (Hs00164004_m1), COL3A1 (Hs00943809_m1), FN1 (Hs01549976_m1), ACTA (Hs00426835_g1), MMP9 (Hs00957562_m1), MMP2 (Hs01548727_m1), MMP1 (Hs00899658_m1), TIMP-1 (Hs99999139_m1), TIMP-2 (Hs00234278_m1), ADAM-12 (Hs01106101_m1), ADAM-17 (Hs01041915_m1), TP53 (Hs01034249_m1).

Techniques: Derivative Assay, Expressing

Journal: Oncotarget

Article Title: FoxM1 promotes breast tumorigenesis by activating PDGF-A and forming a positive feedback loop with the PDGF/AKT signaling pathway

doi:

Figure Lengend Snippet: (A) Correlation of FoxM1 and PDGF-A expression in different breast cancer cells revealed by Western blot analysis. (B) 4T07 cells and BT474 cells were transfected with pcDNA3.1-FoxM1 or pcDNA3.1 vector for 48 hours. Cell lysates were collected for Western blot analysis of FoxM1 and PDGF-A (upper panel). Relative mRNA levels of FoxM1 and PDGF-A were detected by quantitative real-time polymerase chain reaction (qRT-PCR) (lower panel). (C) 4T1 cells and MDA-MB-231 cells were transfected with SiFoxM1 or Sicontrol (50 nM) for 48 hours, and FoxM1 and PDGF-A protein or mRNA levels were detected by Western blot analysis (upper panel) and qRT-PCR (lower panel), respectively. The qRT-PCR results are expressed as percentage of FoxM1 or PDGF-A expression in the experiment groups versus the control groups. All experiments were performed three times. * P < 0.05.

Article Snippet: The membranes were incubated with antibodies against FoxM1 (sc-500, Santa Cruz Biotechnology), PDGF-A (NBP1-19781, Novus Biologicals), PDGFR-A (sc-338, Santa Cruz Biotechnology), phospho-PDGFRA (Tyr 754) (sc-12911, Santa Cruz Biotechnology), AKT (#9272, Cell Signaling Technology), phospho-AKT (Ser473) (#9271, Cell Signaling Technology). β-actin was used as a loading control.

Techniques: Expressing, Western Blot, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Oncotarget

Article Title: FoxM1 promotes breast tumorigenesis by activating PDGF-A and forming a positive feedback loop with the PDGF/AKT signaling pathway

doi:

Figure Lengend Snippet: (A) Diagram shows the sequence and position of two putative FoxM1-binding elements in the PDGF-A promoter. Abbreviations: TSS, transcriptional start site; WT, wild type; Mut, mutant type. (B) Left panel, 4T07 cells were cotransfected with the PDGF-A promoter reporter, pRL-TK, and pcDNA3.1-FoxM1 or pcDNA 3.1; right panel, MDA-MB-231 cells were cotransfected with the PDGF-A promoter reporter, pRL-TK, and SiFoxM1 or Sicontrol (50 nM). Forty-eight hours after transfection, the cells were collected, and the relative PDGF-A promoter activities were measured. Data are from three independent assays. * P < 0.05. (C) Upper panel, the MEF/3T3 Tet-Off cells expressing FoxM1 were treated with doxycycline and hydrochloride (Dox), and the protein levels of FoxM1 and PDGF-A were detected by Western blot analysis; middle panel, MEF/3T3 Tet-Off cells expressing FoxM1 were transfected with the PDGF-A promoter reporter or the control reporter and then treated with Dox for 8 hours. Cells were lysed 48 hours after transfection, and the relative PDGF-A promoter activities were measured. * P < 0.05. (D) Reporter plasmids harboring the wild-type PDGF-A promoter or the corresponding mutant promoter in the FoxM1-binding sites were transfected into MDA-MB-231 cells, and the relative promoter activities were measured as above. Data are from three independent assays. * P < 0.05; ** P < 0.01. (E) Upper panel, electrophoresis mobility shift assay (EMSA) results show the in vitro binding of FoxM1 with PDGF-A promoter. Probes harboring the potential FoxM1-binding sites (site 1 or site 2) were labeled with γ-p 32 ATP and then were incubated with the MDA-MB-231 nuclear extract. For the supershift assay, FoxM1 antibody (1 μg) was added to the reaction. s, shift; ss, super shift. Lower panel, the chromatin immunoprecipitation (ChIP) assay results show the in vivo binding of FoxM1 to the PDGF-A promoter. MDA-MB-231 cell lysis was immunoprecipitated using an anti-FoxM1 antibody or immunoglobulin G. The resulting samples were subjected to semiquantitative polymerase chain reaction using the site-specific primers.

Article Snippet: The membranes were incubated with antibodies against FoxM1 (sc-500, Santa Cruz Biotechnology), PDGF-A (NBP1-19781, Novus Biologicals), PDGFR-A (sc-338, Santa Cruz Biotechnology), phospho-PDGFRA (Tyr 754) (sc-12911, Santa Cruz Biotechnology), AKT (#9272, Cell Signaling Technology), phospho-AKT (Ser473) (#9271, Cell Signaling Technology). β-actin was used as a loading control.

Techniques: Sequencing, Binding Assay, Mutagenesis, Transfection, Expressing, Western Blot, Electrophoresis, Mobility Shift, In Vitro, Labeling, Incubation, Chromatin Immunoprecipitation, In Vivo, Lysis, Immunoprecipitation, Polymerase Chain Reaction

Journal: Oncotarget

Article Title: FoxM1 promotes breast tumorigenesis by activating PDGF-A and forming a positive feedback loop with the PDGF/AKT signaling pathway

doi:

Figure Lengend Snippet: (A) Left panel, MDA-MB-231-shFoxM1 stable cells were treated with PDGF-AA for 24 hours, and the protein levels of FoxM1, PDGF-A, p-AKT, and total AKT were analyzed by Western blot analysis. Right panel, cell proliferation of MDA-MB-231-shFoxM1 treated with or without PDGF-AA was analyzed by XTT assay. (B) Left panel, 4T07-FoxM1 stable cells were treated with LY294002 for 4 hours, and cell lysis was subjected to Western blot analysis using the indicated antibodies. Right panel, cell proliferation of 4T07-FoxM1 stable cells treated with or without LY294002 was analyzed by XTT assay. Data are from three independent experiments. * P < 0.05. (C) 4T07 stable cells with or without FoxM1 overexpression (1×10 5 per mouse) were injected into the mammary fat pad of nude mice (n = 5 for each group), and the tumor diameter (mm) was examined at different time points (left panel). The formed tumors were sectioned, and expression of FoxM1, PDGF-A, and phospho-AKT (p-AKT) was detected by immunohistochemical analysis (right panel). (D) MDA-MB-231 stable cells (5×10 6 per mouse) with or without shRNA-mediated depletion of FoxM1 were injected into the mammary fat pad of nude mice (n = 10 for each group). The tumor diameter was examined at different time points (left panel). The expression of FoxM1, PDGF-A, and p-AKT was analyzed by immunohistochemical analysis (right panel).

Article Snippet: The membranes were incubated with antibodies against FoxM1 (sc-500, Santa Cruz Biotechnology), PDGF-A (NBP1-19781, Novus Biologicals), PDGFR-A (sc-338, Santa Cruz Biotechnology), phospho-PDGFRA (Tyr 754) (sc-12911, Santa Cruz Biotechnology), AKT (#9272, Cell Signaling Technology), phospho-AKT (Ser473) (#9271, Cell Signaling Technology). β-actin was used as a loading control.

Techniques: Western Blot, XTT Assay, Lysis, Over Expression, Injection, Expressing, Immunohistochemical staining, shRNA

Journal: Oncotarget

Article Title: FoxM1 promotes breast tumorigenesis by activating PDGF-A and forming a positive feedback loop with the PDGF/AKT signaling pathway

doi:

Figure Lengend Snippet: (A) BT474 cells were cotransfected with reporter construct harboring the consensus FoxM1 responsive element and pRL-TK and then were treated with PDGF-AA (50 μg/L) or PDGF-AA plus LY294002 (25 μM) for 24 hours. Forty-eight hours later, cells were lysed, and the relative luciferase activity was measured. Data are from three independent assays. * P ≤ 0.05. (B) BT474 cells were treated with PDGF-AA for 24 hours, and the expression of FoxM1, PDGF-A, phospho-AKT (p-AKT), and total AKT was analyzed by Western blotting. (C) MDA-MB-231 cells were transfected with PDGF-A siRNA or control siRNA (50 nM), and the expression of FoxM1, PDGF-A, p-AKT, and total AKT was analyzed by Western blotting. (D and E) BT474 cells were treated with PDGF-AA (50 μg/L) or PDGF-AA plus LY294002 (25 μM) for 24 hours. The expression of FoxM1 was detected by real-time PCR (D) and western blotting (E). (F) BT474 cell proliferation was analyzed by XTT assay after cells were treated with PDGF-AA or PDGF-AA plus LY294002. Data are from three independent assays. * P <0.05.

Article Snippet: The membranes were incubated with antibodies against FoxM1 (sc-500, Santa Cruz Biotechnology), PDGF-A (NBP1-19781, Novus Biologicals), PDGFR-A (sc-338, Santa Cruz Biotechnology), phospho-PDGFRA (Tyr 754) (sc-12911, Santa Cruz Biotechnology), AKT (#9272, Cell Signaling Technology), phospho-AKT (Ser473) (#9271, Cell Signaling Technology). β-actin was used as a loading control.

Techniques: Construct, Luciferase, Activity Assay, Expressing, Western Blot, Transfection, Real-time Polymerase Chain Reaction, XTT Assay

Journal: Oncotarget

Article Title: FoxM1 promotes breast tumorigenesis by activating PDGF-A and forming a positive feedback loop with the PDGF/AKT signaling pathway

doi:

Figure Lengend Snippet: (A) Upper panel, expression of FoxM1, PDGF-A, and p-AKT was examined by immunohistochemical assay in 67 breast cancer specimens and 10 adjacent normal tissues. Images shown are representatives of the examined samples. Magnification 200×. Lower panel, the level of FoxM1, PDGF-A, and p-AKT staining was scored from 0 to 12. The expression of FoxM1, PDGF-A, and p-AKT between non-tumor (N) and tumor specimens (T) were evaluated. ** P < 0.01. (B) The expression correlations among FoxM1, PDGF-A, and p-AKT were significant as determined by Pearson's correlation test. r : Pearson correlation coefficient. (C) Schematic diagram shows the mechanism underlying FoxM1, PDGF-A, and AKT: FoxM1 transcriptionally activates PDGF-A and the AKT signaling pathway; in return, the activated AKT pathway upregulates FoxM1 expression, which forms a positive feedback loop and promotes breast cancer cell growth and tumorigenesis.

Article Snippet: The membranes were incubated with antibodies against FoxM1 (sc-500, Santa Cruz Biotechnology), PDGF-A (NBP1-19781, Novus Biologicals), PDGFR-A (sc-338, Santa Cruz Biotechnology), phospho-PDGFRA (Tyr 754) (sc-12911, Santa Cruz Biotechnology), AKT (#9272, Cell Signaling Technology), phospho-AKT (Ser473) (#9271, Cell Signaling Technology). β-actin was used as a loading control.

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Cell Death & Disease

Article Title: Nintedanib decreases muscle fibrosis and improves muscle function in a murine model of dystrophinopathy

doi: 10.1038/s41419-018-0792-6

Figure Lengend Snippet: Fibroblasts proliferation was analyzed after 2, 4, and 6 days of culture ( a–d ). We cultured fibroblast with increasing concentrations of nintedanib in a culture containing 10% fetal bovine serum (FBS) and we observed decreased fibroblast proliferation after 4 and 6 days in a dose-dependent manner ( a ). Fibroblasts were cultured in the presence of platelet-derived growth factor AA (PDGF-AA, 10 ng/mL) ( b ), basic fibroblast growth factor (FGFb, 10 ng/mL),( c) or vascular endothelial growth factor A (VEGF-A, 50 ng/mL) ( d ) with 1% FBS with or without nintedanib 0.4 μM. Only PDGF-AA increased cell proliferation, an effect that was reversed with the addition of nintedanib to the culture. Fibroblast migration was analyzed using a scratch assay. Nintedanid treatment at 0.4 μM reverted the promigratory effect of PDGF-AA, FGFb, VEGF, and CTGF ( e ). Representative images of this assay are shown in ( f ). Nintedanib treatment at 0.4 μM (blue bars) reverted the effect of PDGF-AA in the expression of ADAM metallopeptidase domain 17 ( ADAM-17) , tissue inhibitor of metalloproteinase 1 (TIMP-1) and tissue inhibitor of metalloproteinase 2 (TIMP-2) ( g ). Nintedanib treatment at 0.4 μM (blue bars) produced a statistically significant reduction of collagen type I alpha 1 chain (COL1A1) , collagen type III alpha 1 chain (COL3A1) , fibronectin 1 ( FN1) , platelet-derived growth factor A (PDGFA) , connective tissue growth factor (CTGF) , transforming growth factor beta 1 (TGFβ1) expression compared with control samples (black bars) analyzed by qPCR ( h ). Data are expressed as means ± SD. N = 3 per group. * P < 0.05, ** P < 0.01, *** P < 0.005

Article Snippet: All mRNA-specific FAM-labeled primers/probe were purchased from Applied Biosystems and detected cDNA from the following genes: Ctgf (Mm01192933_g1), Pdgfa (Mm01205760_m1), Pdgfb (Mm00440677_m1), Tgfb1 (Mm01178820_m1), Col1a1 (Mm00801666_g1), Col3a1 (Mm01254476_m1), Fn1 (Mm01256744_m1), Adgre1 (Mm00802529_m1), PDGFA (Hs00964426_m1), CTGF (Hs01026927_g1), PDGFB (Hs00966522_m1), TGFB1 (Hs00998133_m1), COL1A1 (Hs00164004_m1), COL3A1 (Hs00943809_m1), FN1 (Hs01549976_m1), ACTA (Hs00426835_g1), MMP9 (Hs00957562_m1), MMP2 (Hs01548727_m1), MMP1 (Hs00899658_m1), TIMP-1 (Hs99999139_m1), TIMP-2 (Hs00234278_m1), ADAM-12 (Hs01106101_m1), ADAM-17 (Hs01041915_m1), TP53 (Hs01034249_m1).

Techniques: Cell Culture, Derivative Assay, Migration, Wound Healing Assay, Expressing, Produced

Journal: Cell Death & Disease

Article Title: Nintedanib decreases muscle fibrosis and improves muscle function in a murine model of dystrophinopathy

doi: 10.1038/s41419-018-0792-6

Figure Lengend Snippet: Collagen type I alpha 1 chain (Col1a1), Collagen type III alpha 1 chain ( Col3a1 ), Fibronectin 1 ( Fn1 ), Platelet-derived growth factor A ( Pdgfa ), Platelet-derived growth factor B ( Pdgfb ), Connective tissue growth factor ( Ctgf ), Transforming growth factor beta 1 ( Tgfβ1 ), and Adhesion G protein-coupled receptor E1 ( Adgre1 ) showed changes in relative abundance following nintedanib in quadriceps ( a–h ), diaphragm ( i–p ), and tibialis anterior ( q–y ). Col1a1, Col3a1, Pdgfa, Tgfb1, and Adgre1 gene expression was increased in mdx mice compared with WT . Col1a1, Col3a1, Pdgfa, Pdgfb, Tgfb1, and Adgre1 expression was reduced in all muscles analyzed from nintedanib-treated mdx mice compared with mdx mice. In contrast, Ctgf expression was increased in muscles from nintedanib-treated mdx mice compared with mdx mice. Data are expressed as means ± SD. Genetic background mouse strain C57BL (WT); n = 5, mdx mice (mdx), n = 5; nintedanib-treated mdx mice (mdx + Ninte), n = 7. * P < 0.05, ** P < 0.01, *** P < 0.005

Article Snippet: All mRNA-specific FAM-labeled primers/probe were purchased from Applied Biosystems and detected cDNA from the following genes: Ctgf (Mm01192933_g1), Pdgfa (Mm01205760_m1), Pdgfb (Mm00440677_m1), Tgfb1 (Mm01178820_m1), Col1a1 (Mm00801666_g1), Col3a1 (Mm01254476_m1), Fn1 (Mm01256744_m1), Adgre1 (Mm00802529_m1), PDGFA (Hs00964426_m1), CTGF (Hs01026927_g1), PDGFB (Hs00966522_m1), TGFB1 (Hs00998133_m1), COL1A1 (Hs00164004_m1), COL3A1 (Hs00943809_m1), FN1 (Hs01549976_m1), ACTA (Hs00426835_g1), MMP9 (Hs00957562_m1), MMP2 (Hs01548727_m1), MMP1 (Hs00899658_m1), TIMP-1 (Hs99999139_m1), TIMP-2 (Hs00234278_m1), ADAM-12 (Hs01106101_m1), ADAM-17 (Hs01041915_m1), TP53 (Hs01034249_m1).

Techniques: Derivative Assay, Expressing