Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Aberrant activation of hedgehog signaling promotes cell proliferation via the transcriptional activation of forkhead Box M1 in colorectal cancer cells

doi: 10.1186/s13046-017-0491-7

Figure Lengend Snippet: FoxM1 and Gli1 are highly expressed in CRC tissues. a The FoxM1 and Gli1 protein expression levels were analyzed in eight pairs of randomly selected human CRC tissues and matched adjacent non-tumor tissues by Western blotting. b Quantification of the Western blot ( a ). Significance was assessed using a paired samples t -test. c Immunohistochemistry staining of FoxM1 and Gli1 protein expression in a representative human CRC sample and the matched adjacent normal tissue sample in the same section. The areas of carcinoma and adjacent tissues are marked. The subcellular locations of Gli1 and FoxM1 are indicated by red arrows. d , e and f The mRNA expression level of FoxM1 analyzed in three published human CRC sample expression profiling studies using the R2 data sheet ( http://r2.amc.nl ). p < 0.05 was considered significant

Article Snippet: Soluble chromatin was immunoprecipitated with 4 μg of anti-Gli1 goat polyclonal antibody (Santa Cruz, sc-6152) or anti-Gli2 goat polyclonal antibody (Santa Cruz, sc-20290), and 4 μg of goat normal IgG (Santa Cruz, sc-2028) was added as a random control.

Techniques: Expressing, Western Blot, Immunohistochemistry, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Aberrant activation of hedgehog signaling promotes cell proliferation via the transcriptional activation of forkhead Box M1 in colorectal cancer cells

doi: 10.1186/s13046-017-0491-7

Figure Lengend Snippet: The expression of FoxM1 is regulated by Hh-Gli1 signaling. a Western blot analysis of the Gli1, FoxM1, and CCNB1 protein levels in six CRC cell lines. b Real-time PCR analysis of the Gli1, FoxM1, and CCNB1 mRNA expression levels in six CRC cell lines. The HT29 cells’ mRNA expression level was used as the normalized control. c Detection of Gli1, FoxM1, and CCNB1 protein expression in Caco2 cells after treatment with Hh signaling pathway activator. Caco2 cells were treated with 1 μM or 2 μM purmorphamine for 48 h, lysed and subjected to a Western blot analysis. d - f The inhibition of the Hh signaling pathway inhibited the protein expression of Gli1, FoxM1, and CCNB1, as demonstrated by the Western blot. HCT116 cells were transfected with Gli1-miRNAi or control miRNAi constructs for 48 h ( d ) or treated with the Gli inhibitor GANT61 ( e ) or Smo inhibitor cyclopamine ( f ) at the indicated time and concentrations. The cells were lysed and subjected to Western blotting. g Real-time PCR analysis of Gli1, FoxM1, and CCNB1 mRNA expression in HCT116 cells after treatment with GANT61 or cyclopamine. The mRNA expression levels were normalized to that of β-actin and expressed as fold change compared with the DMSO control. Error bars represent the mean and S.D. of three independent experiments. miR-Gli1: miRNAi-Gli1. *, p < 0.05; **, p < 0.01

Article Snippet: Soluble chromatin was immunoprecipitated with 4 μg of anti-Gli1 goat polyclonal antibody (Santa Cruz, sc-6152) or anti-Gli2 goat polyclonal antibody (Santa Cruz, sc-20290), and 4 μg of goat normal IgG (Santa Cruz, sc-2028) was added as a random control.

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Control, Inhibition, Transfection, Construct

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Aberrant activation of hedgehog signaling promotes cell proliferation via the transcriptional activation of forkhead Box M1 in colorectal cancer cells

doi: 10.1186/s13046-017-0491-7

Figure Lengend Snippet: Gli1 transactivates the FoxM1 promoter. a Schematic diagram of four potential Gli1 binding sites (BS1, BS2, BS3, and BS4) in the FoxM1 promoter. The 9-base pair sequence of the Gli1 binding site and the sequences of four Gli1 binding sites identified in the FxoM1 promoter are shown. b Chromatin was isolated from HT29 cells, and ChIP assays were performed with goat IgG control, Gli1-specific and Gli2-specific antibodies. DM, DNA marker. c Schematic diagram of a series of FoxM1-luciferase constructs. BS4-Mut, binding site 4 mutation; △BS4, binding site 4 deletion. d FoxM1 luciferase constructs, as indicated, were transfected into LoVo cells together with full-length V5-Gli1 plasmid or control vector for 48 h and subjected to a luciferase reporter assay. The results were normalized to the Renilla luciferase activity and are expressed as the fold change in relative luciferase activity compared with the control. Error bars represent the mean and S.D. of three independent experiments. **, p < 0.01

Article Snippet: Soluble chromatin was immunoprecipitated with 4 μg of anti-Gli1 goat polyclonal antibody (Santa Cruz, sc-6152) or anti-Gli2 goat polyclonal antibody (Santa Cruz, sc-20290), and 4 μg of goat normal IgG (Santa Cruz, sc-2028) was added as a random control.

Techniques: Binding Assay, Sequencing, Isolation, Control, Marker, Luciferase, Construct, Mutagenesis, Transfection, Plasmid Preparation, Reporter Assay, Activity Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Aberrant activation of hedgehog signaling promotes cell proliferation via the transcriptional activation of forkhead Box M1 in colorectal cancer cells

doi: 10.1186/s13046-017-0491-7

Figure Lengend Snippet: Gli1 regulates CRC cell proliferation depending on FoxM1. a - d ) LoVo cells were separately or simultaneously treated with 1 μM purmorphamine and 1 μM thiostrepton for the indicated time. a The Gli1, FoxM1, and CCNB1 protein expression levels were examined by immunoblotting after drug treatment for 48 h. b Cell viability was detected after 6 days using an MTT assay. c LoVo cells treated with indicated drugs were cultured for 2 weeks, and outgrowth colonies were stained with crystal violet. d The matched colony count of ( c ). Error bars represent the mean and S.D. of three independent experiments. **, p < 0.01. e and f The cell cycle profile of LoVo cells was examined by fluorescence-activated cell sorting (FACS) with propidium iodine (PI) staining after 48 h of drug treatment ( e ), and the percentages of multinucleate cells were quantified and are shown as a histogram ( f ). Purm: purmorphamine; Thios: thiostrepton. Error bars represent the mean and S.D. of three independent experiments. *, p < 0.05

Article Snippet: Soluble chromatin was immunoprecipitated with 4 μg of anti-Gli1 goat polyclonal antibody (Santa Cruz, sc-6152) or anti-Gli2 goat polyclonal antibody (Santa Cruz, sc-20290), and 4 μg of goat normal IgG (Santa Cruz, sc-2028) was added as a random control.

Techniques: Expressing, Western Blot, MTT Assay, Cell Culture, Staining, Fluorescence, FACS

Journal:

Article Title: FoxF1 and FoxL1 Link Hedgehog Signaling and the Control of Epithelial Proliferation in the Developing Stomach and Intestine * S⃞

doi: 10.1074/jbc.M808103200

Figure Lengend Snippet: Gli1 occupies its site in the Foxf1 promoter in intestinal chromatin. A, schematic representation of the Foxf1/Foxl1 locus. Physical genomic distances are relative to the Foxl1 gene. The binding site assayed here is located in region B. B, quantification of real-time PCR analysis of the enrichment of the Foxf1 promoter element at position -46.9 relative to the transcriptional start site (TSS) of Foxl1. QPCR was performed on five input samples and five ChIP samples, and the enrichment was calculated relative to a non-target gene (myelin basic protein). (**) p value <0.01 according to Student's t test. Data represent the mean ± S.E. C, representative agarose gel of the ChIP experiment showing the presence of the Foxf1 promoter fragment in the Gli1 ChIP but not the IgG control ChIP.

Article Snippet: Chromatin was immunoprecipitated with a polyclonal goat anti-Gli1 antibody (Santa Cruz Biotechnology, antibody sc-6153).

Techniques: Binding Assay, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Control

Journal:

Article Title: FoxF1 and FoxL1 Link Hedgehog Signaling and the Control of Epithelial Proliferation in the Developing Stomach and Intestine * S⃞

doi: 10.1074/jbc.M808103200

Figure Lengend Snippet: Foxf1 and Foxl1 expression are dependent on Gli2 and Gli3, in vivo. A, prospective stomach and intestine were dissected from day 12.5 days postcoital embryos. RT-PCR for Barx1 confirms that our stomach anlage is expressing this gastric marker. Cdx2 expression is strong in the intestine, as expected. Some Cdx2 expression is also present in the stomach fraction, indicating that these samples also contained some prospective duodenal tissue. B and C, Foxf1, Foxl1, and Gli1 mRNA levels in E12.5 stomach (B) and intestine (C) anlage in Gli2 and Gli3 compound mutants, as assessed by quantitative RT-PCR. Tissues from 3-5 embryos were assayed for each genotype. Wild type (WT) levels are set equal to 1.0. (*) p value <0.05 according to Student's independent t test. Data represent the mean ± S.E.

Article Snippet: Chromatin was immunoprecipitated with a polyclonal goat anti-Gli1 antibody (Santa Cruz Biotechnology, antibody sc-6153).

Techniques: Expressing, In Vivo, Reverse Transcription Polymerase Chain Reaction, Marker, Quantitative RT-PCR

Journal:

Article Title: FoxF1 and FoxL1 Link Hedgehog Signaling and the Control of Epithelial Proliferation in the Developing Stomach and Intestine * S⃞

doi: 10.1074/jbc.M808103200

Figure Lengend Snippet: Foxf1 and Foxl1 are induced in gut mesenchyme upon administration of Shh. E14.5 mesenchyme from stomach (Stom) and intestine (Intest) were cultured with 0.5% bovine serum albumin (vehicle (Veh), set equal to 1.0) or recombinant Shh-N for 72 h, and Foxf1 and Foxl1 mRNA levels were assessed by quantitative RT-PCR (A). Rel, relative. Gli1 induction by Shh-N is also shown (B). Data are representative of two experiments, each performed in triplicate. (*) p value <0.05 or <0.01 (**) according to Student's independent t test. Data represent the mean ± S.E.

Article Snippet: Chromatin was immunoprecipitated with a polyclonal goat anti-Gli1 antibody (Santa Cruz Biotechnology, antibody sc-6153).

Techniques: Cell Culture, Recombinant, Quantitative RT-PCR

Journal: Bangladesh Journal of Pharmacology

Article Title: Ursolic acid benzaldehyde chalcone leads to inhibition of cell proliferation and arrests cycle in G1/G0 phase in ovarian cancer

doi: 10.3329/bjp.v10i2.21753

Figure Lengend Snippet: Figure 5: (A) UABC blocks hedgehog signalling, GLI1 expression of rhabdomyosarcoma cell lines. The RMS cell lines were lysed and the expression of GLI1 and β-actin was detected by western blot analysis. For hedgehog signalling dependency, the RMS cells were treated for 48 hours with UABC. GLI1, GLI2, PTCH1, and IGF2 mRNA expression from untreated and treated RMS cells was measured by quantitative real-time PCR in relation to the house-keeping gene TBP as a calibrator. (B) Hedgehog pathway activity. The RMS-13 cells were transiently transfected with 900 ng hedgehog-responsive reporter plasmid (p11 x Gli) or control plasmid (pGL3-TK) and treated for 24 hours with UABC and 10 μM cyclopamine or vehicle. Reporter assay experiments were repeated three times and transfections performed in duplicate

Article Snippet: Incubation of membranes with mouse anti-human caspase-3, rabbit antihuman cleaved caspase-3, rabbit antihuman β-actin (Cell Signalling Technology, Danvers, MA, USA), mouse anti-human COX4, rabbit antihuman cytochrome c (Clontech, Mountain View, CA, USA) or goat antihuman GLI1 (Santa Cruz Biotechnology, Santa Figure1: Structure of ursolic acid benzaldehyde chalcone Cruz, CA, USA) antibodies was performed for 2 hours.

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Activity Assay, Transfection, Plasmid Preparation, Control, Reporter Assay

Journal: Bangladesh Journal of Pharmacology

Article Title: Ursolic acid benzaldehyde chalcone leads to inhibition of cell proliferation and arrests cycle in G1/G0 phase in ovarian cancer

doi: 10.3329/bjp.v10i2.21753

Figure Lengend Snippet: Figure 6: Hedgehog activation assay. The NIH-3T3 cells were transiently transfected with 1 μg sonic hedgehog (Shh) expression plasmid. For the inhibition of hedgehog signalling, cells were additionally exposed to UABC and 10 μM cyclopamine (CHX.) after 48 hours transfection. Expression of the murine genes SHT, Gli1, Gli2, Ptch1, and Igf2 was determined after an incubation period of 48 hours using quantitative real-time PCR in relation to the house-keeping gene TBP as a calibrator

Article Snippet: Incubation of membranes with mouse anti-human caspase-3, rabbit antihuman cleaved caspase-3, rabbit antihuman β-actin (Cell Signalling Technology, Danvers, MA, USA), mouse anti-human COX4, rabbit antihuman cytochrome c (Clontech, Mountain View, CA, USA) or goat antihuman GLI1 (Santa Cruz Biotechnology, Santa Figure1: Structure of ursolic acid benzaldehyde chalcone Cruz, CA, USA) antibodies was performed for 2 hours.

Techniques: Activation Assay, Transfection, Expressing, Plasmid Preparation, Inhibition, Incubation, Real-time Polymerase Chain Reaction

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Fibrinogen inhibits sonic hedgehog signaling and impairs neonatal cerebellar development after blood-brain barrier disruption.

doi: 10.1073/pnas.2323050121

Figure Lengend Snippet: Fig. 4. Fibrinogen depletion rescues cerebellar pathology in systemic neonatal inflammation. (A) Schematic of LPS-induced neonatal inflammation. Created with BioRender.com. Image of P10 cerebellum with nuclei (blue) labeled with DAPI to show representative cerebellar cross-sectional area (dotted white line) quantified in B and image areas (solid white square) shown in C and D. (B–D) Fga+/+, LPS-treated littermate controls. Fga−/−, LPS-treated fibrinogen knockout mice. Data are mean ± SEM from n = 9 (Fga+/+) and 7 to 8 (Fga−/−) mice per group. ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, Mann–Whitney. (B) Cerebellar weight and cross-sectional area at P10. (C) SHH activation marker GLI1 (red), Purkinje cell marker CALB (green), and nuclei (blue, DAPI) in P10 cerebellum. Dotted white line outlines the EGL. (Scale bar, 50 µm.) (D) Proliferation marker phospho-histone H3 (green) and nuclei (blue, DAPI) in P10 cerebellum. Dotted white line outlines the EGL. (Scale bar, 50 µm.)

Article Snippet: Primary antibodies for IF were CALB (1:200, rabbit monoclonal, #13176, Cell Signaling Technology), fibrinogen (1:1,000, rabbit polyclonal, gift from J. Degen, Cincinnati), GLI1 (1:100, goat polyclonal, #AF3455, R&D Systems), phosphoHistone H3 (1:250, rabbit monoclonal, #53348, Cell Signaling Technology), IBA1 (1:500, rabbit polyclonal, #019- 19741 Wako), and VCAM1 (1:100, rat monoclonal, #550547, BD Pharmingen).

Techniques: Labeling, Knock-Out, MANN-WHITNEY, Activation Assay, Marker

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Fibrinogen inhibits sonic hedgehog signaling and impairs neonatal cerebellar development after blood-brain barrier disruption.

doi: 10.1073/pnas.2323050121

Figure Lengend Snippet: Fig. 3. Fibrinogen inhibits the SHH pathway and proliferation of CGNPs. (A) Schematic of SHH effects in CGNPs and fibrinogen inhibition. (B) GLI1, MYCN, and CCND1 protein levels 24 h after control or SHH (3 µg/mL) treatment of primary CGNPs in the presence of fibrinogen (2.5 mg/mL). Blot representative of n = 2 independent experiments. Δ, densitometry fold change values. (C) Mitosis marker phospho-histone H3 (green) and nuclei (blue, DAPI) 48 h after control or SHH (3 µg/mL) treatment of primary CGNPs in the presence of fibrinogen (0.1, 0.3, 0.9, 1.5, 2.5 mg/mL). (Scale bar, 50 µm.) Data are mean ± SEM from n = 3 independent experiments. ***P < 0.001, ****P < 0.0001, one-way ANOVA with Dunnett. (D) GLI1, MYCN, and CCND1 protein levels in cerebellum 24 h after IC injection of aCSF or fibrinogen (2 µL, 5 mg/mL) at P2. Densitometry fold change values are mean ± SEM from n = 3 mice per condition. *P < 0.05, unpaired t test. Created with BioRender.com. (E) SHH activation marker GLI1 (red) and nuclei (blue, DAPI) in cerebellum 24 h after IC injection of aCSF or fibrinogen (2 µL, 5 mg/mL) at P2. (Scale bar, 100 µm.) Data are mean ± SEM from n = 5 mice per group. **P < 0.01, Mann–Whitney.

Article Snippet: Primary antibodies for IF were CALB (1:200, rabbit monoclonal, #13176, Cell Signaling Technology), fibrinogen (1:1,000, rabbit polyclonal, gift from J. Degen, Cincinnati), GLI1 (1:100, goat polyclonal, #AF3455, R&D Systems), phosphoHistone H3 (1:250, rabbit monoclonal, #53348, Cell Signaling Technology), IBA1 (1:500, rabbit polyclonal, #019- 19741 Wako), and VCAM1 (1:100, rat monoclonal, #550547, BD Pharmingen).

Techniques: Inhibition, Control, Marker, Injection, Activation Assay, MANN-WHITNEY