ereg ab  (R&D Systems)

Journal: Cell death & disease

Article Title: mTORC1 signaling pathway integrates estrogen and growth factor to coordinate vaginal epithelial cells proliferation and differentiation.

doi: 10.1038/s41419-022-05293-8

Figure Lengend Snippet: Fig. 6 EREG acts as a potential factor for vaginal epithelial cell proliferation and differentiation. A Heatmap of the expression levels of ErbB signaling pathway signature genes in the vagina of control and Rptor cKO mice in the presence or absence of E2 administration. B qPCR was performed to verify the genes in the frame in A. n = 7 (OVX control mice), n = 5 (OVX Rptor cKO mice), n = 6 (OVX control mice treated with E2), n = 5 (OVX Rptor cKO mice treated with E2). Values are expressed as the mean ± SEM. C Representative images of the immunofluorescence staining of EREG in the vagina in OVX control and Rptor cKO mice administrated with or without E2. Nuclei were stained with DAPI. Microscopy with magnification ×20. Scale bars: 75 μm. D OVX control and Rptor cKO mice were administrated with E2 and/or EREG. The vaginas were harvested for PAS staining, Ki67 immunofluorescence staining, and TUNEL staining. In PAS staining, nuclei were stained with hematoxylin. Scale bars: 50 μm. In Ki67 immunofluorescence staining and TUNEL staining, nuclei were stained with DAPI. Microscopy with magnification ×20. Scale bars: 75 μm. E Expression levels of Krt6a, Krt6b, Krt10, Krt13, Krt16 in the vagina of the mice were quantified using qPCR. n = 6 (OVX control mice treated with E2), n = 5 (OVX Rptor cKO mice treated with E2), n = 4 (OVX Rptor cKO mice treated with E2 and EREG). Values are expressed as the mean ± SEM. F The vaginas as indicated in D were harvested for immunofluorescence staining of YAP1, nuclei were stained with DAPI. n = 6 (OVX control mice treated with E2), n = 5 (OVX Rptor cKO mice treated with E2), n = 4 (OVX Rptor cKO mice treated with E2 and EREG). The experiments were repeated three times. Microscopy with magnification ×20. Scale bars: 75 μm.

Article Snippet: For immunofluorescence staining, sections were incubated with the following primary antibodies: anti-Raptor (sc-81537, Santa Cruz Biotechnology), anti-Phospho-S6 Ribosomal Protein (Ser235/236) (62016, Cell Signaling Technology), anti-PR (9856 S, Cell Signaling Technology), anti-ERalpha Ab (ab32063, Abcam), anti-Ki67 Ab (ab15580, Abcam), anti-YAP1 Ab (13584-1-AP, Proteintech), anti-EREG Ab (MAB1068, R&D Systems) overnight at 4 °C.

Techniques: Expressing, Control, Staining, Microscopy, TUNEL Assay

Journal: Cell death & disease

Article Title: mTORC1 signaling pathway integrates estrogen and growth factor to coordinate vaginal epithelial cells proliferation and differentiation.

doi: 10.1038/s41419-022-05293-8

Figure Lengend Snippet: Fig. 8 A possible scheme depicting the role of mTORC1 signaling in estrogen-induced epithelial cell proliferation and differentia- tion in mouse vagina. A mTORC1 signaling participates in the proliferation and differentiation of vaginal epithelium by promoting the expression level of PR and EREG-YAP1 in Rptorfl/flmice. B Loss of Rptor compromises the estrogen-induced proliferation and differ- entiation of vaginal epitheliums through down-regulating the expression of PR and EREG.

Article Snippet: For immunofluorescence staining, sections were incubated with the following primary antibodies: anti-Raptor (sc-81537, Santa Cruz Biotechnology), anti-Phospho-S6 Ribosomal Protein (Ser235/236) (62016, Cell Signaling Technology), anti-PR (9856 S, Cell Signaling Technology), anti-ERalpha Ab (ab32063, Abcam), anti-Ki67 Ab (ab15580, Abcam), anti-YAP1 Ab (13584-1-AP, Proteintech), anti-EREG Ab (MAB1068, R&D Systems) overnight at 4 °C.

Techniques: Expressing

Journal: Theranostics

Article Title: EREG-driven oncogenesis of Head and Neck Squamous Cell Carcinoma exhibits higher sensitivity to Erlotinib therapy

doi: 10.7150/thno.47176

Figure Lengend Snippet: High EREG expression predicts a poor prognosis in HNSCC patients. (A) The microarray analysis between HN4 and HOK cells. (B) RT-PCR analysis of EREG mRNA levels in HOK and five other HNSCC cell lines. (C) Densitometric EREG mRNA data in B were normalized to GAPDH mRNA levels. (D) EREG expression from 7 paired cases of fresh-frozen HNSCC tumors was examined by Western blotting. (E) Densitometric EREG protein data in D were normalized to GAPDH protein levels. Significant differences were detected using a Wilcoxon signed-rank test (P < 0.005) in EREG expression between adjacent normal oral tissues and cancer tissues. (F) Representative images of EREG expression in normal tissues and HNSCC tissues via immunohistochemical (IHC) staining. (G) IHC scores of EREG expression in HNSCC tissues (n = 80) and paired adjacent normal tissues (n = 80). Significant differences were detected (P < 0.005) in EREG expression between adjacent normal tissues and HNSCC tissues. (H) EREG gene mutations in HNSCC tissues according to the cBioPortal for Cancer Genomics. (I) High EREG expression significantly correlates with the poor survival rate of HNSCC patients. The survival rates of patients with EREG-positive and EREG-negative tumors (P< 0.01) were determined using the Kaplan-Meier survival test

Article Snippet: The endogenous activity of each sample was blocked in a 3% hydrogen peroxide/PBS, avidin/biotin solution (Invitrogen, CA, USA), after which the samples were incubated with a polyclonal goat anti-human EREG antibody (R&D Systems, MN, USA) overnight at 4°C.

Techniques: Expressing, Microarray, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunohistochemical staining, Immunohistochemistry

Journal: Theranostics

Article Title: EREG-driven oncogenesis of Head and Neck Squamous Cell Carcinoma exhibits higher sensitivity to Erlotinib therapy

doi: 10.7150/thno.47176

Figure Lengend Snippet: EREG promotes HNSCC tumorigenicity in vitro and in vivo . (A) Effect of EREG on the cell growth of 3D-cultured HNSCC cancer cell lines. Three HNSCC cell lines were seeded on day 0 and cultured in 3D conditions through day 12. Representative images of each cell line were captured on days 2, 4, 6, 8, 10 and 12. Each cell line was treated with or without EREG from day 1 through day 11. Scale bars indicate 100 µm. (B) The growth of 3D-cultured CAL27, HN6 and HN13 cells treated with or without EREG was analyzed. Each data point represents the mean value and standard deviation of 3 replicate wells. (C) HN4 cells stably transfected with control or EREG-specific shRNAs were injected into nude mice. Tumor growth was monitored every 3 days; tumor size and weight were recorded. The data are presented as the mean ± SEM from five mice. *P < 0.05 and **P < 0.01 for vector control cells compared with their EREG-knockdown clones. (D) HN6 cells stably transfected with control or Flag-EREG constructs were injected into nude mice. Tumor growth was monitored every 3 days, and tumor size and weight were recorded. The data are presented as the mean ± SEM from five mice. *P < 0.05 and **P < 0.01 for the vector control cells compared with the Flag-EREG clones.

Article Snippet: The endogenous activity of each sample was blocked in a 3% hydrogen peroxide/PBS, avidin/biotin solution (Invitrogen, CA, USA), after which the samples were incubated with a polyclonal goat anti-human EREG antibody (R&D Systems, MN, USA) overnight at 4°C.

Techniques: In Vitro, In Vivo, Cell Culture, Standard Deviation, Stable Transfection, Transfection, Control, Injection, Plasmid Preparation, Knockdown, Clone Assay, Construct

Journal: Theranostics

Article Title: EREG-driven oncogenesis of Head and Neck Squamous Cell Carcinoma exhibits higher sensitivity to Erlotinib therapy

doi: 10.7150/thno.47176

Figure Lengend Snippet: EREG associates with EGFR and triggers EGFR signaling. (A) Immunoblot (IB) of HN13 and HN6 cells treated with epiregulin (50 ng/ml) at the indicated time points and probed with an anti-phosphotyrosine (p-Tyr) antibody. (B) IB of SACC and HNSCC cancer cells treated with epiregulin (50 ng/ml) for 5 min and probed with an anti-p-Tyr antibody. (C) Human phospho-RTK antibody array analysis of HN6 cells serum starved for 24 hr, followed by epiregulin (50 ng/mL) treatment for 5 min. (D) IB of HN13 (left) and HN6 (right) cells treated with epiregulin (50 ng/ml) at various time points. (E) Immunofluorescence staining for EGFR in HN6 cells treated with or without 50 ng/mL epiregulin.

Article Snippet: The endogenous activity of each sample was blocked in a 3% hydrogen peroxide/PBS, avidin/biotin solution (Invitrogen, CA, USA), after which the samples were incubated with a polyclonal goat anti-human EREG antibody (R&D Systems, MN, USA) overnight at 4°C.

Techniques: Western Blot, Ab Array, Immunofluorescence, Staining

Journal: Theranostics

Article Title: EREG-driven oncogenesis of Head and Neck Squamous Cell Carcinoma exhibits higher sensitivity to Erlotinib therapy

doi: 10.7150/thno.47176

Figure Lengend Snippet: EREG triggers EGFR downstream signaling in an EGFR kinase-dependent manner. (A) HN13 and HN6 cells pretreated with erlotinib and AG1478 followed by epiregulin treatment and IB with the indicated antibodies. (B) IB of HN6 and HN13 cells transfected with individual small interfering RNAs (siRNAs) against EGFR in the presence or absence of epiregulin (50 ng/ml). (C) IB of HN6 and HN12 cells treated with or without epiregulin (50 ng/ml). (D) Human phosphokinase antibody array analysis of HN6 cells treated with or without epiregulin (50 ng/ml) for 5 min. (E) IB of HN13 (left) and HN6 (right) cells treated with epiregulin (50 ng/ml) at different time points. (F) HN6 and HN13 cells pretreated with erlotinib and AG1478 followed by epiregulin treatment and immunoblotting (IB) with the indicated antibodies.

Article Snippet: The endogenous activity of each sample was blocked in a 3% hydrogen peroxide/PBS, avidin/biotin solution (Invitrogen, CA, USA), after which the samples were incubated with a polyclonal goat anti-human EREG antibody (R&D Systems, MN, USA) overnight at 4°C.

Techniques: Transfection, Ab Array, Western Blot

Journal: Theranostics

Article Title: EREG-driven oncogenesis of Head and Neck Squamous Cell Carcinoma exhibits higher sensitivity to Erlotinib therapy

doi: 10.7150/thno.47176

Figure Lengend Snippet: EREG binds to EGFR via N57 and requires the EGFR domains I and III. (A) HEK293 cells were transiently coexpressed with FLAG-EREG and HA-tagged EGFR. Cell extracts were immunoprecipitated separately with anti-FLAG or anti-HA antibodies, and the associated EGFR and EREG proteins were examined by Western blotting. (B) Endogenous EGFR and EREG were immunoprecipitated from HN4 cells, and bound endogenous EREG and EGFR were examined by Western blotting. (C) The cellular location of EGFR (red) and EREG (green) was examined by immunofluorescence staining (nuclei were stained with DAPI; blue). Scale bar, 50 μm. (D) Schematic diagram of the WT, domain I deletion (∆D1), and domain III mutation (D355T/F357A) constructs of EGFR (FLAG-EGFR-ECD). The numbers represent amino acid residues. (E-F) FLAG-tagged WT or deletion mutants of EGFR were coexpressed with HA-EREG in HEK293 cells. Extracts were immunoprecipitated with an anti-FLAG or anti-HA antibody, and bound EREG or EGFR was examined by Western blotting using the anti-HA or anti-FLAG antibody (for input control, see Figure S4C ). (G) Sequence alignment of EREG from different species. (H) Schematic diagram of various EREG NQ mutants used in this study. The numbers indicate amino acid positions on the EREG. (I-J) HA-tagged WT or NQ mutants of EREG were coexpressed with FLAG-EGFR in HEK293 cells. EREG and EGFR were immunoprecipitated with anti-HA and anti-FLAG antibodies, respectively, and analyzed by Western blotting.

Article Snippet: The endogenous activity of each sample was blocked in a 3% hydrogen peroxide/PBS, avidin/biotin solution (Invitrogen, CA, USA), after which the samples were incubated with a polyclonal goat anti-human EREG antibody (R&D Systems, MN, USA) overnight at 4°C.

Techniques: Immunoprecipitation, Western Blot, Immunofluorescence, Staining, Mutagenesis, Construct, Control, Sequencing

Journal: Theranostics

Article Title: EREG-driven oncogenesis of Head and Neck Squamous Cell Carcinoma exhibits higher sensitivity to Erlotinib therapy

doi: 10.7150/thno.47176

Figure Lengend Snippet: EREG-induced C-Myc expression is required for EREG-promoted oncogenesis in HNSCC. (A) EREG-related gene enrichment analysis from TCGA using the cBio Cancer Genomics Portal. (B) Western blot analysis for C-Myc from three different HNSCC cell lines treated with 50 ng/mL epiregulin as indicated. (C) Western blot analysis of EREG and C-Myc expression in HN4 and HN30 cells after transfection with siEREG or siNC siRNAs. (D) RT-PCR analysis of C-Myc mRNA levels in HN13 and HN6 cells treated with 50 ng/mL epiregulin as indicated. (E) CAL27 and HN13 cells were pretreated with various inhibitors for 1 h followed by stimulation with epiregulin for 2 h. The level of C-Myc was examined by Western blot analysis. (F) 3D culture of HN6 cells treated with or without epiregulin and BET BD inhibitors. Scale bar=100 μm. (G) A correlation was found between EREG and C-Myc at the mRNA level in four gene expression data sets.

Article Snippet: The endogenous activity of each sample was blocked in a 3% hydrogen peroxide/PBS, avidin/biotin solution (Invitrogen, CA, USA), after which the samples were incubated with a polyclonal goat anti-human EREG antibody (R&D Systems, MN, USA) overnight at 4°C.

Techniques: Expressing, Western Blot, Transfection, Reverse Transcription Polymerase Chain Reaction, Gene Expression

Journal: Theranostics

Article Title: EREG-driven oncogenesis of Head and Neck Squamous Cell Carcinoma exhibits higher sensitivity to Erlotinib therapy

doi: 10.7150/thno.47176

Figure Lengend Snippet: EREG-induced C-Myc expression depends on EGFR activity. (A) Western blot analysis of C-Myc, p-EGFR, and EGFR from tumor cell lines pretreated with various EGFR inhibitors for 1 h followed by stimulation with epiregulin for 2 h. (B) OncoPrint of EREG-EGFR-MYC pathway alterations in HNC. Genomic alterations of different members of this pathway are mutually exclusive. (C) Western blot analysis of C-Myc, p-EGFR, EGFR, p-AKT, AKT, p-ERK, ERK, p-STAT3, and STAT3 from CAL27 and HN6 cells pretreated with various inhibitors for 1 h followed by stimulation with epiregulin for 2 h.

Article Snippet: The endogenous activity of each sample was blocked in a 3% hydrogen peroxide/PBS, avidin/biotin solution (Invitrogen, CA, USA), after which the samples were incubated with a polyclonal goat anti-human EREG antibody (R&D Systems, MN, USA) overnight at 4°C.

Techniques: Expressing, Activity Assay, Western Blot

Journal: Theranostics

Article Title: EREG-driven oncogenesis of Head and Neck Squamous Cell Carcinoma exhibits higher sensitivity to Erlotinib therapy

doi: 10.7150/thno.47176

Figure Lengend Snippet: EGFR-Erk activation by epiregulin is sustained. (A) Representative time courses of EGFR phosphorylation at Y1086 in SACC-83 cells induced by EGF, EREG, AREG or TGF-α. An anti-EGFR antibody was used as a loading control. (B) Representative time courses of EGFR phosphorylation at Y1173 in CAL27 cells induced by EGF, EREG, AREG or TGF-α. An anti-EGFR antibody was used as a loading control. (C-D) Quantification of EGFR phosphorylation time courses, normalized by the signal at 5 min. The data are plotted on the same graph for multiple independent experiments quantitating phosphorylation at Y1068 and Y1173. (E-F) Representative time courses of Erk phosphorylation in SACC-83 and CAL27 cells induced by different EGFR ligands. (G-H) Quantification of Erk phosphorylation time courses, normalized by the signal at 5 min. The data are plotted on the same graph for multiple independent experiments quantifying Erk phosphorylation.

Article Snippet: The endogenous activity of each sample was blocked in a 3% hydrogen peroxide/PBS, avidin/biotin solution (Invitrogen, CA, USA), after which the samples were incubated with a polyclonal goat anti-human EREG antibody (R&D Systems, MN, USA) overnight at 4°C.

Techniques: Activation Assay, Phospho-proteomics, Control

Journal: Theranostics

Article Title: EREG-driven oncogenesis of Head and Neck Squamous Cell Carcinoma exhibits higher sensitivity to Erlotinib therapy

doi: 10.7150/thno.47176

Figure Lengend Snippet: High EREG expression predicted a better sensitivity to erlotinib treatment in HNSCC. (A) Cell extracts were prepared from 6 HNC cell lines, which were divided into two groups based on EREG expression, and EREG expression was analyzed by Western blotting. (B-C) Colony formation was assessed in many cancer cell lines based on EREG expression levels with erlotinib (2 µM) treatment. The number of colonies was calculated. (D-E) Effect of erlotinib on the cell growth of 3D-cultured HN4 and HN12 cells. High-EREG-expressing HN4 and low-EREG-expressing HN12 cell lines were seeded on day 0 and cultured in 3D conditions through day 12. Representative images of each cell line were captured on days 2 and 12. Each cell line was treated with or without erlotinib from day 1 through day 11. Scale bars indicate 100 µm. (F-G) Colony-formation analysis was performed on SACC-83 and HN30 cells. The low-EREG-expressing SACC-83 cells were treated with erlotinib, epiregulin or erlotinib plus epiregulin, and the high-EREG-expressing HN30 cells stably transfected with control or EREG-specific shRNAs were treated with or without erlotinib. The number of colonies was calculated. (H-I) Effect of erlotinib on the cell growth of 3D-cultured high-EREG-expressing HN30 cells stably transfected with control or EREG-specific shRNAs. Scale bars indicate 100 µm. * P<0.05. (J-K) Effect of epiregulin and erlotinib on the cell growth of 3D-cultured CAL27 cells. Low-EREG-expressing CAL27 cell lines were seeded on day 0 and cultured in 3D conditions through day 12. Representative images of each cell line were captured on days 2 and 12. Each cell line was treated with epiregulin, erlotinib or epiregulin plus erlotinib from day 1 through day 11. Scale bars indicate 100 µm. (L-O) HN30 cells stably transfected with control or EREG-specific shRNAs were injected into nude mice followed by treatment with or without erlotinib. Tumor growth was monitored every 3 days; tumor size and weight were recorded. The data are presented as the mean ± SEM from five mice.

Article Snippet: The endogenous activity of each sample was blocked in a 3% hydrogen peroxide/PBS, avidin/biotin solution (Invitrogen, CA, USA), after which the samples were incubated with a polyclonal goat anti-human EREG antibody (R&D Systems, MN, USA) overnight at 4°C.

Techniques: Expressing, Western Blot, Cell Culture, Stable Transfection, Transfection, Control, Injection

Journal: Theranostics

Article Title: EREG-driven oncogenesis of Head and Neck Squamous Cell Carcinoma exhibits higher sensitivity to Erlotinib therapy

doi: 10.7150/thno.47176

Figure Lengend Snippet: The proposed model showing that EGFR domains I and III and the N57 residue of EREG are required for EREG-induced EGFR-Erk-C-Myc signaling activation, which in turn promotes oncogenesis and increases erlotinib sensitivity in HNSCC patients.

Article Snippet: The endogenous activity of each sample was blocked in a 3% hydrogen peroxide/PBS, avidin/biotin solution (Invitrogen, CA, USA), after which the samples were incubated with a polyclonal goat anti-human EREG antibody (R&D Systems, MN, USA) overnight at 4°C.

Techniques: Residue, Activation Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Temporal expression of growth factors triggered by epiregulin regulates inflammation development.

doi: 10.4049/jimmunol.1400562

Figure Lengend Snippet: FIGURE 2. Areg, BTC, TGF-a, and FGF2 enhance the expressions of IL-6 and chemokines via the PI3K/NF-kB axis. (A) IL-6 (1 mg) and IL-17 (1 mg) were injected into the left ankle joints of F759 mice on days 0, 1, and 2. Immunohistochemistry of the left ankle joints was performed by using Abs against Areg, epiregulin (Ereg), FGF2, TGF-a, p-STAT3, p-p65, p-EGFR, type 1 collagen, and vimentin on day 7. These experiments were performed at least three times independently. Frequency of cells that showed activation of the inflammation amplifier (p-STAT3+p-p65+), received EGFR signaling (p-EGFR+), or produced growth factors (Areg+Ereg+FGF2+TGF-a+) is indicated. Col1, type 1 collagen; Vim, vimentin. *p , 0.05, **p , 0.01 (Student t test). (B) BC1 cells were stimulated with human IL-6 plus soluble IL-6Ra and/or mouse IL-17 for 24 h with or without Areg, BTC, TGF-a, FGF2, PLGF2, and TNC. Culture supernatants were collected and assessed using ELISA specific for IL-6. Samples without growth factors (filled columns) were compared with samples with each growth factor. xp , 0.05, *p , 0.01, #,†p , 0.001 (one-way ANOVA). (C and D) mRNA expressions of IL-6 (C) and CCL20 (D) in BC1 cells 3 h after stimulation with human IL-6 plus soluble IL-6Ra and mouse IL-17 with or without Areg, BTC, TGF-a, and FGF2 were evaluated using real- time PCR. Samples without growth factors (filled columns) were compared with samples with each growth factor. *p , 0.05, **p , 0.01, ***p , 0.001 (one-way ANOVA). (E) BC1 cells were stimulated with human IL-6 plus soluble IL-6Ra in the presence or absence of Areg, BTC, TGF-a, or FGF2 for 24 h with or without 0.5 h pretreatment of LY294002 (3 mM) or DMSO vehicle control. Culture supernatants were collected and assessed using ELISA specific for mouse IL-6. Cell survival was evaluated based on mitochondrial activity. *p , 0.05, **p , 0.01, ***p , 0.001 (Student t test). (F) BC1 cells were stimulated with human IL-6 plus soluble IL-6Ra and mouse IL-17 in the presence or absence of Areg (A), BTC (B), TGF-a (T), or FGF2 (F) for 30 min and then investigated for the phosphorylation of Akt and p65. (G) IL-6 and IL-17 were injected into the ankle joints of NF-kB reporter Tg/F759 mice with or without 0.2 mg Areg, BTC, TGF-a, or FGF2 followed by analysis of NF-kB reporter activity in the ankle joints on day 7 using the luciferase reporter assay system. *p , 0.05 (one-way ANOVA). Mean scores 6 SD (A–E) and mean scores 6 SEM (G) are shown.

Article Snippet: The following Abs were used for in vivo neutralization and immunohistochemistry: monoclonal anti-mouse Areg Ab, anti-mouse BTC Ab, anti- mouse epiregulin Ab, anti-human TGF-a Ab, anti-mouse PLGF2 Ab, anti-human/mouse TNC Ab (R&D Systems, Minneapolis, MN), antimouse FGF2 Ab (Millipore, Tokyo, Japan), polyclonal anti-mouse epiregulin Ab (Santa Cruz Biotechnology, Santa Cruz, CA), anti-mouse FGF2 Ab (Abcam, Tokyo, Japan), and purified rat IgG (Sigma-Aldrich, Tokyo, Japan).

Techniques: Injection, Immunohistochemistry, Activation Assay, Produced, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Control, Activity Assay, Phospho-proteomics, Luciferase, Reporter Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Temporal expression of growth factors triggered by epiregulin regulates inflammation development.

doi: 10.4049/jimmunol.1400562

Figure Lengend Snippet: FIGURE 3. Presence of epiregulin-triggered temporal expressions in affected tissues of cytokine-induced arthritis. (A) mRNA expressions of Areg, Btc, Tgfa, epiregulin (Ereg), Fgf2, Plgf2, and Tnc in BC1 cells in the presence or absence of stimulation with IL-17 and IL-6 were evaluated 3 h later using real- time PCR. (B) mRNA expressions of Areg, Btc, Tgfa, Ereg, Fgf2, Plgf2, and Tnc in BC1 cells in the presence or absence of epiregulin stimulation were evaluated 3 h later using real-time PCR. (C) IL-17 (0.2 mg) and IL-6 (0.2 mg) on days 0, 1, and 2 were injected into the knee joints of F759 mice followed by analysis of expressions of Ereg, Areg, and TGF-a in joint synovial tissues on days 0, 1, 3, 5, and 7 (n = 3 for each condition). (D and E) IL-17 (0.2 mg) and IL-6 (0.2 mg) on days 0, 1, and 2 were injected into the knee joints of F759 mice in the presence or absence of joint injections of anti-Ereg Ab (1 mg, n = 12), anti-Areg Ab (1 mg, n = 12), anti-BTC Ab (1 mg, n = 12), anti–TGF-a Ab (1 mg, n = 12), anti-FGF2 Ab (1 mg, n = 12), anti-PLGF2 Ab (1 mg, n = 12), anti-TNC Ab (1 mg, n = 12), or control IgG (1 mg, n = 12) on days 0, 1, 2, 4, and 6 followed by analysis of the expressions of Ereg, Areg, Btc, TGF-a, FGF2, Plgf2, and Tnc (D) and Ereg (E) in joint synovial tissues on day 7. (F–I) IL-6 (1 mg) and IL-17 (1 mg) on days 0, 1, and 2 were injected into the left ankle joints of F759 mice followed staining by using antibodies against Ereg, TGF-a, and FGF2 in paraffin sections of left ankle joints on days 1 (F) and 7 (H) by immunohistochemistry. These experiments were performed at least three times independently; representative data are shown. Arrows indicate cells expressing growth factors in the ankle joint synovial tissues. Scale bars, 100 mm. Quantification of the histological analysis (10 3 0.1 mm2 field) for (F) and (H) is shown (G and I). Mean scores 6 SD (A–E) and mean scores 6 SEM (G and I) are shown. The p values were calculated using a Student t test (A and B) and one-way ANOVA (D and E). *p , 0.05, **p , 0.01, ***p , 0.001.

Article Snippet: The following Abs were used for in vivo neutralization and immunohistochemistry: monoclonal anti-mouse Areg Ab, anti-mouse BTC Ab, anti- mouse epiregulin Ab, anti-human TGF-a Ab, anti-mouse PLGF2 Ab, anti-human/mouse TNC Ab (R&D Systems, Minneapolis, MN), antimouse FGF2 Ab (Millipore, Tokyo, Japan), polyclonal anti-mouse epiregulin Ab (Santa Cruz Biotechnology, Santa Cruz, CA), anti-mouse FGF2 Ab (Abcam, Tokyo, Japan), and purified rat IgG (Sigma-Aldrich, Tokyo, Japan).

Techniques: Real-time Polymerase Chain Reaction, Injection, Control, Staining, Immunohistochemistry, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Temporal expression of growth factors triggered by epiregulin regulates inflammation development.

doi: 10.4049/jimmunol.1400562

Figure Lengend Snippet: FIGURE 4. Growth factors are critical for the development of an MS model, EAE. (A) Serum concentrations of Areg, BTC, TGF-a, FGF2, PLGF, and TNC in patients suffering from MS (n = 21) compared with healthy age- and sex-matched subjects (n = 15). (B) mRNA expressions of epiregulin (Ereg), Areg, Btc, TGF-a, FGF2, Plgf2, and Tnc in the L5 cord 7 d after transfer of pathogenic CD4+ T cells were evaluated using real-time PCR. (C–E) Pathogenic CD4+ T cells isolated from EAE mice were i.v. transferred into wild-type C57BL/6 mice in the presence or absence of anti–TGF-a Ab administration (i.p., days 0–5 after the pathogenic CD4+ T cell transfer). (C) Clinical EAE scores (n = 5 each) and (D) serum IL-6 concentrations in mice (n = 15). (E) Mononuclear cells from L5 spinal cords of Th17-transferred C57BL/6 mice were isolated on day 10. The resulting cell populations were counted and stimulated in vitro with MOG peptide and bone marrow–derived dendritic cells. Twenty-four hours after in vitro stimulation, intracellular IL-17 and IFN-g levels were examined. The numbers of CD4+IL-17+ and CD4+IFN-g+ T cells in spinal cords were significantly lower in recipients treated with anti–TGF-a Ab (n = 5) than in those treated with control IgG (n = 5). Individual scores, mean scores (A), and mean scores 6 SEM (B–E) are shown. The p values were calculated using Wilcoxon tests (A and C) and Student t test (B, D, and E). *p , 0.05, **p , 0.01, ***p , 0.001.

Article Snippet: The following Abs were used for in vivo neutralization and immunohistochemistry: monoclonal anti-mouse Areg Ab, anti-mouse BTC Ab, anti- mouse epiregulin Ab, anti-human TGF-a Ab, anti-mouse PLGF2 Ab, anti-human/mouse TNC Ab (R&D Systems, Minneapolis, MN), antimouse FGF2 Ab (Millipore, Tokyo, Japan), polyclonal anti-mouse epiregulin Ab (Santa Cruz Biotechnology, Santa Cruz, CA), anti-mouse FGF2 Ab (Abcam, Tokyo, Japan), and purified rat IgG (Sigma-Aldrich, Tokyo, Japan).

Techniques: Real-time Polymerase Chain Reaction, Isolation, In Vitro, Derivative Assay, Control