Journal: PLoS Genetics

Article Title: Binding of TFIIIC to SINE Elements Controls the Relocation of Activity-Dependent Neuronal Genes to Transcription Factories

doi: 10.1371/journal.pgen.1003699

Figure Lengend Snippet: (A and B) Changes in H3K9K14ac at c-Fos (A) and Gadd45b (B) loci. From the top, gene locus structure (blue), SINEs (shades of gray) with c-Fos RSINE1 (A) and Gadd45b B1F (B) highlighted, H3K9K14ac tag density per 100 bp in control conditions (CTR) and in response to NEE (NEE), and −log(p-value) of regions of statistical difference in H3K9K14ac levels are shown. Tag density profiles are not directly comparable, as they have not been normalized across samples. (C) Exposure to NEE increased histone H3 acetylation at c-Fos RSINE1 and Gadd45b B1F and not at TSSs. Adult mice were either exposed to NEE for 45 min or left untreated, somatosensory cortex was dissected and subjected to ChIP using either H3K9K14ac or histone H3 antibodies, followed by qPCR. Histograms show the ratio of immunoprecipitation efficiency between H3K9K14ac and H3 antibodies relative to total chromatin input (average and s.e.m. of at least 6 mice per condition are shown; *, P<0.05, Student's t-test). (D and E) Mouse somatosensory cortex (D) and primary cortical neurons (E) were subjected to Gtf3c1 ChIP followed by qPCR targeting c-Fos RSINE1 and Gadd45b B1F . Histograms show the efficiency of immunoprecipitation relative to total chromatin input, expressed as percentage of total input. 5S rRNA gene locus was used as positive control. Jdp2 B1 , Gapdh B4 and a genomic region devoid of SINEs and H3K9K14ac ChIPseq tags (ctrl) were used as negative control (average and s.e.m. of at least 3 experiments are shown; *, P<0.05, Student's t-test). (F) Gtf3c1 binding at c-Fos RSINE1 and Gadd45b B1F increased in response to depolarization. Mouse primary cortical neurons were either exposed to 50 mM KCl for 45 min or left untreated, and subjected to ChIP using Gtf3c1 antibodies, followed by qPCR. Histograms show the immunoprecipitation efficiency of Gtf3c1 and control IgG antibodies relative to total chromatin input. Five previously identified c-Fos enhancers showed no significant Gtf3c1 binding (average and s.e.m. of 3 experiments are shown; *, P<0.05, Student's t-test). (G) p300 was recruited to c-Fos RSINE1 in response to depolarization. Mouse primary cortical neurons were either exposed to 50 mM KCl for 45 min or left untreated, and subjected to ChIP using p300 antibodies, followed by qPCR. Histograms show the immunoprecipitation efficiency of Gtf3c1 antibodies relative to control IgG (average and s.e.m. of 3 experiments are shown; *, P<0.05, Student's t-test).

Article Snippet: Antibodies used were Gtf3c1 (NB100-60657, Novus Biologicals), Gtf3c2 (A301-236A, Bethyl Laboratories), Gtf3c3 (A301-238A, Bethyl Laboratories), Gtf3c4 (A301-239A, Bethyl Laboratories), Gtf3c5 (A301-242A, Bethyl Laboratories), Gtf3c6 (ab107804, Abcam), c-Myc (sc56634, Santa Cruz), Hsp90 (sc1055, Santa Cruz), Gapdh (ab9494, Abcam).

Techniques: Control, Immunoprecipitation, Positive Control, Negative Control, Binding Assay

Journal: Journal of Histochemistry and Cytochemistry

Article Title: Enhanced Expression of Fibroblast Growth Factor Receptor 3 IIIc Promotes Human Esophageal Carcinoma Cell Proliferation

doi: 10.1369/0022155415616161

Figure Lengend Snippet: RT-PCR Analysis and Immunohistochemical Analysis of FGFR3IIIc Expression in Esophageal Squamous Cell Carcinoma.

Article Snippet: A recombinant human FGFR3IIIc Fc chimera (#766-FR-050, lot.CWZ0713081; R&D Systems, Minneapolis, MN), which contains the extracellular region of FGFR3IIIc and Fc region of human IgG 1 , was incubated with anti-FGFR3IIIc antibody (cat#MAB7662, lot.GHK0411041; R&D Systems) in PBS containing 0.1% Tween-20, 3% BSA without γ-globulin (Wako; Kyoto, Japan), and 3% horse serum for 1 hr at room temperature.

Techniques: Immunohistochemical staining, Expressing

Journal: Journal of Histochemistry and Cytochemistry

Article Title: Enhanced Expression of Fibroblast Growth Factor Receptor 3 IIIc Promotes Human Esophageal Carcinoma Cell Proliferation

doi: 10.1369/0022155415616161

Figure Lengend Snippet: Enhanced expression of FGFR3IIIc was associated with proliferating esophageal carcinoma (EC) cells. (A) Immunofluorescence staining of FGFR3IIIc (a, d) and SCC-112 (b, e) in non-cancerous mucosa (NCM; N, non-tumor) and esophageal squamous cell carcinoma (ESCC) (T, tumor), which was diagnosed as stage 0. In confocal microscopic images, strong staining of FGFR3IIIc (red) was observed in EC cells (d) but not in normal esophageal epithelium cells (a). FGFR3IIIc-positive cells in ESCC were consistent with SCC-112-positive cells (green) (d, e, f: white arrows). Nuclei were stained with DAPI (blue). (B) Immunofluorescence staining of Ki-67 (red; a, d) and SCC-112 (green; b, e) in NCM (N) and ESCC (T). The strong staining of Ki-67 was observed in EC cells, and Ki67-positive cells were consistent with SCC-112-positive cells in ESCC (d, e, f: white arrows). On the other hand, staining of Ki-67 and SCC-112 were not observed in NCM (a, b, c). (C) Immunofluorescence staining of DAPI (a, d), FGFR3IIIc (b), Ki-67 (e) and merged images (c, f) in ESCC samples. The expression of FGFR3IIIc was detected in the same cells, which also expressed Ki-67 in consecutive sections (c, f; white arrows). Scale, 20 μm.

Article Snippet: A recombinant human FGFR3IIIc Fc chimera (#766-FR-050, lot.CWZ0713081; R&D Systems, Minneapolis, MN), which contains the extracellular region of FGFR3IIIc and Fc region of human IgG 1 , was incubated with anti-FGFR3IIIc antibody (cat#MAB7662, lot.GHK0411041; R&D Systems) in PBS containing 0.1% Tween-20, 3% BSA without γ-globulin (Wako; Kyoto, Japan), and 3% horse serum for 1 hr at room temperature.

Techniques: Expressing, Immunofluorescence, Staining

Journal: Journal of Histochemistry and Cytochemistry

Article Title: Enhanced Expression of Fibroblast Growth Factor Receptor 3 IIIc Promotes Human Esophageal Carcinoma Cell Proliferation

doi: 10.1369/0022155415616161

Figure Lengend Snippet: Expression of FGFR3IIIc was enhanced in esophageal carcinoma cells diagnosed as stage IA (A, B, and C), stage IB (D and E), stage IIIA (F, G and H), but not in normal esophageal epithelium cells. FGFR3IIIc was not stained by anti-FGFR3IIIc antibody after pre-adsorption with the recombinant human FGFR3IIIc Fc chimera (H). Hematoxylin and eosin staining (H&E stain, A, D, and F), FGFR3IIIc immunostaining (B, E, and G), and SCC-112 immunostaining (C). Strong staining for FGFR3IIIc was observed in esophageal carcinoma cells (B, E, and G: white arrows), and FGFR3IIIc expression was clearly restricted in the carcinoma area, with a clear border. Normal esophageal epithelium cells did not express FGFR3IIIc (B, E, and G: black arrows). FGFR3IIIc-positive cells were consistent with SCC-112-positive cells in the nuclei on consecutive sections (C: white arrows). Scale, 20 μm.

Article Snippet: A recombinant human FGFR3IIIc Fc chimera (#766-FR-050, lot.CWZ0713081; R&D Systems, Minneapolis, MN), which contains the extracellular region of FGFR3IIIc and Fc region of human IgG 1 , was incubated with anti-FGFR3IIIc antibody (cat#MAB7662, lot.GHK0411041; R&D Systems) in PBS containing 0.1% Tween-20, 3% BSA without γ-globulin (Wako; Kyoto, Japan), and 3% horse serum for 1 hr at room temperature.

Techniques: Expressing, Staining, Adsorption, Recombinant, Immunostaining

Journal: Journal of Histochemistry and Cytochemistry

Article Title: Enhanced Expression of Fibroblast Growth Factor Receptor 3 IIIc Promotes Human Esophageal Carcinoma Cell Proliferation

doi: 10.1369/0022155415616161

Figure Lengend Snippet: PCR Primer Sequences.

Article Snippet: A recombinant human FGFR3IIIc Fc chimera (#766-FR-050, lot.CWZ0713081; R&D Systems, Minneapolis, MN), which contains the extracellular region of FGFR3IIIc and Fc region of human IgG 1 , was incubated with anti-FGFR3IIIc antibody (cat#MAB7662, lot.GHK0411041; R&D Systems) in PBS containing 0.1% Tween-20, 3% BSA without γ-globulin (Wako; Kyoto, Japan), and 3% horse serum for 1 hr at room temperature.

Techniques: Sequencing

Journal: Journal of Histochemistry and Cytochemistry

Article Title: Enhanced Expression of Fibroblast Growth Factor Receptor 3 IIIc Promotes Human Esophageal Carcinoma Cell Proliferation

doi: 10.1369/0022155415616161

Figure Lengend Snippet: Enhanced expression of FGFR3IIIc accelerated cell proliferation. (A) Cell proliferation was significantly lower (by 15%) in EC-GI-10 cells treated with FGFR3 siRNA (siFGFR3) than in EC-GI-10 cells treated with sicontrol (sicontrol) after 5 days in culture. Cell proliferation in FGFR3IIIc-overexpressed siFGFR3-EC-GI-10 cells (FGFR3IIIc) was significantly higher than that in siFGFR3-EC-GI-10 cells (by 1.3-fold), whereas the overexpression of FGFR3IIIb (FGFR3IIIb) did not significantly affect cell proliferation rates. Cell proliferation was significantly lower (~25%) in EGFP-infected siFGFR3-EC-GI-10 cells (EGFP) than in siFGFR3-EC-GI-10 cells. The parental EC-GI-10 cells were not treated with siRNA (-) and not infected (-). Cell numbers are the mean ± S.E.M. The experiment was performed with n=6 samples, and repeated thrice. (#, p<0.05 versus the sicontrol cells and *, p<0.05 versus the siFGFR3 cells; t-test, N.S., not significant). (B) Western blotting shows FGFR3 expression levels of parental cells and cells treated with sicontrol, EGFP, siFGFR3, FGFR3IIIb, or FGFR3IIIc. β-actin was expressed equally among the cells. (C) RT-PCR shows that FGF2 was endogenously expressed in parental EC-GI-10 cells.

Article Snippet: A recombinant human FGFR3IIIc Fc chimera (#766-FR-050, lot.CWZ0713081; R&D Systems, Minneapolis, MN), which contains the extracellular region of FGFR3IIIc and Fc region of human IgG 1 , was incubated with anti-FGFR3IIIc antibody (cat#MAB7662, lot.GHK0411041; R&D Systems) in PBS containing 0.1% Tween-20, 3% BSA without γ-globulin (Wako; Kyoto, Japan), and 3% horse serum for 1 hr at room temperature.

Techniques: Expressing, Over Expression, Infection, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of Histochemistry and Cytochemistry

Article Title: Enhanced Expression of Fibroblast Growth Factor Receptor 3 IIIc Promotes Human Esophageal Carcinoma Cell Proliferation

doi: 10.1369/0022155415616161

Figure Lengend Snippet: FGFR3IIIb and FGFR3IIIc Expression in Esophageal Carcinoma (EC) and Non-Cancerous Mucosa (NCM).

Article Snippet: A recombinant human FGFR3IIIc Fc chimera (#766-FR-050, lot.CWZ0713081; R&D Systems, Minneapolis, MN), which contains the extracellular region of FGFR3IIIc and Fc region of human IgG 1 , was incubated with anti-FGFR3IIIc antibody (cat#MAB7662, lot.GHK0411041; R&D Systems) in PBS containing 0.1% Tween-20, 3% BSA without γ-globulin (Wako; Kyoto, Japan), and 3% horse serum for 1 hr at room temperature.

Techniques: Expressing

Journal: Journal of Histochemistry and Cytochemistry

Article Title: Enhanced Expression of Fibroblast Growth Factor Receptor 3 IIIc Promotes Human Esophageal Carcinoma Cell Proliferation

doi: 10.1369/0022155415616161

Figure Lengend Snippet: Gene expression of FGFR3 isoforms in esophageal carcinoma (EC) and non-cancerous mucosa (NCM). Total RNA was extracted from the specimens of EC patients. The gene expression of FGFR3 in 16 patients (E1 to E16) was analyzed by RT-PCR. FGFR3IIIc expression was clearly detected in the EC (T; tumor) of 12 patients (E2, E3, E4, E5, E6, E7, E8, E10, E13, E14, E15, and E16) and in the NCM (N; non-tumor) of 6 patients (E1, E3, E10, E13, E14, and E15). FGFR3IIIb expression was clearly detected in the EC (T) of 12 patients and in the NCM (N) of 11 patients. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: A recombinant human FGFR3IIIc Fc chimera (#766-FR-050, lot.CWZ0713081; R&D Systems, Minneapolis, MN), which contains the extracellular region of FGFR3IIIc and Fc region of human IgG 1 , was incubated with anti-FGFR3IIIc antibody (cat#MAB7662, lot.GHK0411041; R&D Systems) in PBS containing 0.1% Tween-20, 3% BSA without γ-globulin (Wako; Kyoto, Japan), and 3% horse serum for 1 hr at room temperature.

Techniques: Gene Expression, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: Vascular Pharmacology

Article Title: Soluble N-cadherin: A novel inhibitor of VSMC proliferation and intimal thickening

doi: 10.1016/j.vph.2015.11.040

Figure Lengend Snippet: QPCR primers.

Article Snippet: Antibodies were used at the following concentrations: N-cadherin (BD Biosciences: 610920, 1:2500), GAPDH (Chemicon: MAB374, 1:5000), and FGFR1 (R&D Systems: MAB658, 1:50).

Techniques: Sequencing

Journal: Vascular Pharmacology

Article Title: Soluble N-cadherin: A novel inhibitor of VSMC proliferation and intimal thickening

doi: 10.1016/j.vph.2015.11.040

Figure Lengend Snippet: FGF-R was essential for the anti-proliferative effect of soluble N-cadherin. a: Proliferation (percentage of BrdU positive human VSMCs) following treatment with SU5402 (an inhibitor of FGFR1) and 100 pM Fc or SNC-Fc, in the presence of bFGF and PDGF for 24 h. * indicates a significant difference from the Fc control, n = 3. b: Proliferation (percentage of BrdU positive human VSMCs) following siRNA for FGFR1 and treatment with 100 pM Fc or SNC-Fc in the presence of bFGF and PDGF for 24 h. * indicates a significant difference from the relevant Fc control, n = 3. c: Proliferation (percentage of BrdU positive human VSMCs) following 24 h incubation with bFGF and PDGF and 100 pM Fc, SNC-Fc or SNC-Fc with either the N-cadherin binding site mutation (SNC-Fc N-cadmut) or FGF-R binding site mutated (SNC-Fc FGFRmut). * indicates a significant difference from the Fc control, n = 3. d: Western blot for Fc-tag in VSMCs overexpressing FGFR1GFP and incubated with Fc/SNC-Fc/SNC-Fc for 1 h, before immunoprecipitation for GFP tagged FGFR and Western blotting (i). Western blot for Fc-tag in VSMCs overexpressing full length N-cadherin and incubated with Fc/SNC-Fc/SNC-Fc FGFR for 1 h, before lysis and Western blotting (ii). e: The percentage of cells with nuclear FGFR following AMAXA transfection with FGFR1 plasmid and treatment with either serum free media (SF), bFGF and PDGF (GF), and 20, 100 or 200 pM Fc or SNC-Fc plus GF. * indicates a significant difference from SF control, # indicates a significant difference from GF and Fc plus GF, n = 3. Representative images to show nuclear translocation of FGFR1 for the 200 pM dose. Scale bars represent 10 mm. Yellow arrowheads indicate cells with nuclear FGFR1 (green), white arrowheads indicate cells with cytoplasmic FGFR1. Nuclei have been psuedocoloured in red. f: Proliferation (percentage of BrdU positive human VSMCs) following transfection with FGFR1(SP/NLS), a constitutively nuclear form of FGFR1, and treatment with 200 pM Fc or SNC-Fc in the presence of bFGF and PDGF for 24 h. * indicates a significant difference from SF, n = 3.

Article Snippet: Antibodies were used at the following concentrations: N-cadherin (BD Biosciences: 610920, 1:2500), GAPDH (Chemicon: MAB374, 1:5000), and FGFR1 (R&D Systems: MAB658, 1:50).

Techniques: Control, Incubation, Binding Assay, Mutagenesis, Western Blot, Immunoprecipitation, Lysis, Transfection, Plasmid Preparation, Translocation Assay