Journal: Bone

Article Title: The reduced osteogenic potential of Nf1 -deficient osteoprogenitors is EGFR-independent

doi: 10.1016/j.bone.2017.10.012

Figure Lengend Snippet: A: Ereg expression in WT and Nf1-deficient mBMSCs (qPCR, n=3). B: Epiregulin protein expression in WT and Nf1-deficient mBMSCs (Western blot, n=3, Right graph: densitometric analysis). C: Egfr expression in WT and Nf1-deficient mBMSCs (qPCR, n=3). D: EGFR protein expression in WT and Nf1 deficient mBMSCs (Western blot, n=3, Right graph: densitometric analysis). E: Level of phosphorylated EGFR (p-EGFR), EGFR and β-actin in A431 cells treated with the conditioned medium (CM) from WT (grey bar) and Nf1-deficient (KO, black bar) mBMSCs in the presence of IgG control or an epiregulin neutralizing antibody (Western blot, n=3, Right graph: densitometric analysis). * and #: p<0.05 between genotypes and treatments, respectively. qPCR gene expression is normalized by Hprt expression.

Article Snippet: Cells were then treated with the conditioned media plus normal goat IgG control (AB-108-C, R&D Systems) or Epiregulin neutralizing antibody (AF1068-SP, R&D Systems) at the final concentration of 0.4 μg/ml.

Techniques: Expressing, Western Blot, Control, Gene Expression

Journal: Bone

Article Title: The reduced osteogenic potential of Nf1 -deficient osteoprogenitors is EGFR-independent

doi: 10.1016/j.bone.2017.10.012

Figure Lengend Snippet: A–B, D–E and G, H: Expression of early osteoblast marker genes (Alpl, Ibsp) in response to EGFR or Epiregulin inhibition during osteogenic differentiation (A–B: AG-1478, D–E: Poziotinib and G, H: epiregulin-neutralizing antibody) in WT and Nf1-deficient (KO) mBMSCs (qPCR, n=3, * and #: p<0.05 between genotypes and treatments, respectively). C, F and I: ALP activity in response to AG-1478, Poziotinib and Anti-Ereg neutralizing antibodies, respectively (n=3, * and #: p<0.05 between genotypes and treatments, respectively). qPCR gene expression is normalized by Hprt expression.

Article Snippet: Cells were then treated with the conditioned media plus normal goat IgG control (AB-108-C, R&D Systems) or Epiregulin neutralizing antibody (AF1068-SP, R&D Systems) at the final concentration of 0.4 μg/ml.

Techniques: Expressing, Marker, Inhibition, Activity Assay, Gene Expression

Journal: Cell reports

Article Title: Disease-association analysis of an inflammation-related feedback loop.

doi: 10.1016/j.celrep.2013.01.028

Figure Lengend Snippet: Figure 2. The Epiregulin-ErbB1 Pathway Activates the IL-6 Amplifier In Vitro (A) IL-6 amplifier activation was suppressed in the absence of FBS. BC1 cells were stimulated with human IL-6 plus soluble IL-6 receptor and/or IL-17 for 24 hr in the absence or presence of 10% FBS. Gray dots indicate cell survival values monitored by TCO reagent. Left: Culture supernatants were collected and assessed using an ELISA specific for mouse IL-6. Cell survival was also evaluated based on mitochondrial activity (gray squares; all points were above the threshold value). Right: Il-6 mRNA expression 3 hr after stimulation was evaluated using real-time PCR. (B) BC1 cells were stimulated with human IL-6 plus soluble IL-6 receptor and/or IL-17 in the absence of FBS for 3 hr. Epiregulin (Ereg) expression was then evaluated using real-time PCR. (C) BC1 cells were stimulated with human IL-6 plus soluble IL-6 receptor and/or IL-17 in the absence of FBS for 48 hr with or without EGF, another ErbB1 ligand. Culture supernatants were collected and assessed using an ELISA specific for epiregulin. (D) BC1 cells were stimulated with epiregulin in the absence of FBS for 3 hr. Epiregulin (Ereg) expression was evaluated using real-time PCR. (E) BC1 cells were stimulated with human IL-6 plus soluble IL-6 receptor and/or IL-17 in the absence of FBS for 3 hr. ErbB1 (Egfr) expression was then evaluated using real-time PCR. (F) BC1 cells were stimulated with human IL-6 plus soluble IL-6 receptor and/or IL-17 in the absence of FBS for 24 hr with or without various concentrations of epiregulin. Culture supernatants were collected and assessed using ELISA specific for IL-6. (G–I) Il-6 (G), Ccl20 (H), and IkBz (I) mRNA expressions in BC1 cells 3 hr (G and H) or 1 hr (I) after stimulation with human IL-6 plus soluble IL-6 receptor and/or IL-17 in the absence of FBS with or without epiregulin were evaluated using real-time PCR. (J) BC1 cells were treated with a lentivirus encoding shRNA specific for epiregulin (Ereg) and cultured in the presence of puromycin. The resulting cells were stimulated with human IL-6 plus soluble IL-6 receptor and/or IL-17 in the absence of FBS for 48 hr. Culture supernatants were collected and assessed using ELISA specific for mouse IL-6. Mean scores ± SD are shown. p values were calculated using Student’s t tests. * and #, p < 0.05; ** and ##, p < 0.01; ***p < 0.001. See also Figure S7 and Table S10.

Article Snippet: The following antibodies were used for in vivo neutralization: monoclonal anti-mouse epiregulin antibody (R&D Systems, Minneapolis) and purified rat IgG (Sigma-Aldrich).

Techniques: In Vitro, Activation Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, Expressing, Real-time Polymerase Chain Reaction, shRNA, Cell Culture

Journal: Cell reports

Article Title: Disease-association analysis of an inflammation-related feedback loop.

doi: 10.1016/j.celrep.2013.01.028

Figure Lengend Snippet: Figure 3. The Epiregulin Signal Enhances NFkB Activation via the PI3K a Pathway (A) MEF cells were stimulated with epiregulin in the presence or absence of IL-6 and IL-17 and then investigated for the phosphorylation of Akt, p65, STAT3, and ERK1/ERK2. (B) HeLa cells were stimulated with epiregulin in the presence or absence of IL-17 and then investigated for NFkB reporter activity using a 53 NFkB-luc construct. (C) BC1 cells were stimulated with epiregulin in the presence or absence of LY294002 and then investigated for NFkB reporter activity using a 53 NFkB-luc construct. (D) IKKa- and IKKg-deficient MEF cells as well as wild-type MEF cells (WT) were stimulated with epiregulin (100 ng/ml) in the presence or absence of human IL-6 plus soluble IL-6 receptor and/or IL-17 for 12 hr. Culture supernatants were collected and assessed using an ELISA specific for mouse IL-6. (E and F) BC1 cells were stimulated with human IL-6 plus soluble IL-6 receptor and/or IL-17 in the absence of FBS with or without epiregulin and various concentrations of PIK75 (E) or TGX221 (F) for 24 hr. Cell survival was also evaluated based on mitochondrial activity. Culture supernatants were collected and assessed using an ELISA specific for mouse IL-6. (G and H) (G) BC1 cells were treated with a lentivirus encoding shRNA specific for Pik3ca and stimulated with epiregulin (100 ng/ml) in the presence or absence of human IL-6 plus soluble IL-6 receptor and/or IL-17 in the absence of FBS for 24 hr. Culture supernatants were collected and assessed using an ELISA specific for mouse IL-6. (H) Pik3ca expression was also evaluated using real-time PCR. Mean scores ± SD are shown. p Values were calculated using Student’s t tests. *p < 0.05; **p < 0.01; ***p < 0.001; NS, not significant. See also Figure S9 and Table S10.

Article Snippet: The following antibodies were used for in vivo neutralization: monoclonal anti-mouse epiregulin antibody (R&D Systems, Minneapolis) and purified rat IgG (Sigma-Aldrich).

Techniques: Activation Assay, Phospho-proteomics, Activity Assay, Construct, Enzyme-linked Immunosorbent Assay, shRNA, Expressing, Real-time Polymerase Chain Reaction

Journal: Cell reports

Article Title: Disease-association analysis of an inflammation-related feedback loop.

doi: 10.1016/j.celrep.2013.01.028

Figure Lengend Snippet: Figure 4. Epiregulin-ErbB1 Signaling Triggers Autoimmune Diseases in Mouse Models (A) IL-6 and IL-17 on days 6, 7, and 8 were injected into the joints of F759 mice in the presence or absence of joint injections of lentivirus encoding shRNA specific for ErbB1 (Egfr) (n = 6), Ereg (n = 6), or a nontarget sequence (n = 6) on days 0, 2, and 4 followed by analysis of epiregulin and IL-6 expression in joint synovial tissues on day 15. (B) Clinical arthritis scores from the left legs of F759 mice after left leg joint injections of 0.1 mg IL-17 and IL-6, respectively, on days 6, 7, and 8 and joint injections of lentivirus encoding shRNA specific for ErbB1 (Egfr) (open squares, open triangles, and crosses; n = 3), NFkB p65 (Rela) (asterisks; n = 3), or a nontarget sequence (diamonds; n = 3) on days 0, 2, and 4. (C) Clinical arthritis scores from the left legs of F759 mice after left leg joint injections of 0.1 mg IL-17 and IL-6, respectively, on days 6, 7, and 8 and joint injections of a lentivirus encoding shRNA specific for epiregulin (Ereg) (crosses, asterisks, and open circles; n = 3), NFkB p65 (Rela) (open triangles; n = 3), or a nontarget sequence (squares; n = 3) on days 0, 2, and 4 and those from the left legs of F759 mice after left leg joint injections of saline alone (filled diamonds, n = 3). (D) Clinical arthritis scores from the left legs of F759 mice after left leg joint injections of 0.1 mg IL-17 and IL-6, respectively, on days 0, 1, and 2 and joint injections of anti-epiregulin antibodies (1 mg) on days 0–23 (open squares, n = 3) and from the left legs of F759 mice after left leg joint injections of IL-17, IL-6, and control IgG (open circles, n = 3), or saline (open triangles, n = 3). (E) Clinical arthritis scores from the left legs of F759 mice after left leg joint injections of 0.1 mg IL-17 and IL-6, respectively, on days 0, 1, and 2 and PD153035 (10 mg) on days 0–23 (triangles, n = 5) and from the left legs of F759 mice after left leg joint injections of IL-17, IL-6, and DMSO (squares, n = 5), or injections of DMSO only (diamonds, n = 5). (F) Clinical arthritis scores from the left legs of F759 mice after left leg joint injections of 0.1 mg IL-17 and IL-6, respectively, on days 0, 1, and 2 and PD168393 (10 mg) on days 0–24 (triangles, n = 5) and from the left legs of F759 mice after left leg joint injections of IL-17, IL-6, and DMSO (squares, n = 5), or injections of DMSO only (diamonds, n = 5). (G) Clinical arthritis scores from the left legs of F759 mice after left leg joint injections of 0.1 mg IL-17 and IL-6, respectively, on days 0, 1, and 2 and gefitinib (10 mg) on days 0–22 (triangles, n = 4) and from the left legs of F759 mice after left leg joint injections of IL-17, IL-6, and DMSO (squares, n = 4), or DMSO only (diamonds, n = 4). (H) Ankle joints from each mouse in (D) were fixed and embedded in paraffin. Histological analysis was performed using sections stained with hematoxylin-eosin. These experiments were performed at least three times independently; representative data are shown. (I) WT mice (2 months old) were intravenously injected with Th17 cells from WT mice with EAE and intraperitoneally injected with gefitinib (open squares, n = 5) or DMSO (open diamonds, n = 5) on days 0–6. (J) WT mice (2 months old) were intravenously injected with Th17 cells from WT mice with EAE and intraperitoneally injected with gefitinib (closed bars, n = 5) or DMSO (open bars, n = 5) on days 0–6. Mononuclear cells from the spinal cords of Th17-transferred C57BL/6 mice were isolated on day 13. The resulting cell populations

Article Snippet: The following antibodies were used for in vivo neutralization: monoclonal anti-mouse epiregulin antibody (R&D Systems, Minneapolis) and purified rat IgG (Sigma-Aldrich).

Techniques: Injection, shRNA, Sequencing, Expressing, Saline, Control, Staining, Isolation

Journal: Cell reports

Article Title: Disease-association analysis of an inflammation-related feedback loop.

doi: 10.1016/j.celrep.2013.01.028

Figure Lengend Snippet: Figure 5. Epiregulin-ErbB1 Signaling Is Involved in Activation of the Human IL-6 Amplifier (A) Human synovial cell lines were stimulated for 3 hr with human IL-6 plus soluble IL-6 receptor and/or IL-17 in the absence of FBS with or without epiregulin (100 ng/ml). Il-6 expression in the resulting cells was evaluated using real-time PCR. (B) Human synovial cells were stimulated for 3 hr with human IL-6 plus soluble IL-6 receptor and/or IL-17 in the presence of FBS with or without PD153035 (10 mg/ml). Il-6 expression in the resulting cells was evaluated using real-time PCR. (C) Human synovial cells were stimulated with human IL-6 plus soluble IL-6 receptor and/or IL-17 or epiregulin in the absence of FBS for 3 hr. Ereg expression in the resulting cells was evaluated using real-time PCR. (D–F) Serum epiregulin concentrations in patients with (D) rheumatoid arthritis (n = 11), (E) atherosclerosis (n = 50), and (F) multiple sclerosis (n = 21) compared with healthy-aged, sex-matched subjects (rheumatoid arthritis, n = 26; atherosclerosis n = 64; multiple sclerosis, n = 15). Mean scores ± SD are shown. p values were calculated using Student’s t tests (* and #, p < 0.05; **p < 0.01; ***p < 0.001). Related to Figures S7, S8, and Table S10.

Article Snippet: The following antibodies were used for in vivo neutralization: monoclonal anti-mouse epiregulin antibody (R&D Systems, Minneapolis) and purified rat IgG (Sigma-Aldrich).

Techniques: Activation Assay, Expressing, Real-time Polymerase Chain Reaction

Journal: Science immunology

Article Title: Epiregulin is a dendritic cell-derived EGFR ligand that maintains skin and lung fibrosis

doi: 10.1126/sciimmunol.abq6691

Figure Lengend Snippet: (A) Sankey diagram of enriched receptor-ligand pairs in SSc skin and at least two lung scRNA-Seq datasets. Ribbon width is proportional to 1/rank of the skin SSc data. (B) Plot of the CellphoneDB ranks (adjusted p-values) of the interaction of EREG with EGFR in our skin scRNA-Seq data as well as our analysis of available data from SSc skin (15) and lung (33, 41, 42). Dotted line shows rank = 0.05. (C) Ereg relative expression during a time course of tissue digestion of healthy mouse skin, n=3 per time point. (D) Expression of EREG in our UMAP embedded scRNA-Seq data. (E) Heatmap of co-expressed genes by SSc EREG-expressing APC (EREG+) compared to healthy EREG+ APC and EREG− APC groups. For clarity, the raw gene list was filtered to genes primarily expressed by immune cells. (F) Expression of FCN1 in our UMAP embedded scRNA-Seq data. (G) Immunofluorescence images of EREG and FCN1 in SSc skin. (H, I) Analysis of EREG expression in SSc compared to healthy controls (H) and compared to modified Rodnan Skin Score (mRSS) (I) using data from (49). (J) Low and high magnification photomicrographs of skin and lung from SSc and healthy subject samples stained with EREG antibody. Dashed boxes delineate region shown in higher magnification image. Arrowheads label positive cells. (K) Enumeration of EREG+ cells in SSc skin dermis and lung (n=3 slides each, skin samples from patients SSc1, 3, and 4, 10 high power fields (hpf) per slide). Slides were imaged with a Keyence BZ-X800 microscope. Low power images are at 10x magnification and stitched together. High power images are 40x magnification. Data are means ± SD (***P<0.001, ****P<0.0001) analyzed with one-way analysis of variance (ANOVA) with Tukey multiple-comparisons test (C) and unpaired two-tailed Student t test (K).

Article Snippet: EREG neutralizing antibody (R&D Systems MAB1068 clone 189611) or mouse IgG2a isotype control (Bio X Cell BE0085) 10 mg/kg diluted in 100 μl PBS was given subcutaneously twice weekly on the dorsal neck of anesthetized mice.

Techniques: Expressing, Immunofluorescence, Modification, Staining, Microscopy, Two Tailed Test

Journal: Science immunology

Article Title: Epiregulin is a dendritic cell-derived EGFR ligand that maintains skin and lung fibrosis

doi: 10.1126/sciimmunol.abq6691

Figure Lengend Snippet: (A) Expression fold change of EREG when THP-1 monocytes were incubated with each indicated cytokine. (B) EREG protein quantification from supernatant of THP-1 incubated with IFNa2 for 4 hours, n=4 per group, data is representative from 2 independent experiments. (C-E) EREG expression fold change from freshly isolated peripheral blood CD14+ monocytes (C) and CD1c+ dendritic cell precursors (D) or cultured human BMDC (E) after incubation with IFNα2. (F) Expression fold change of NOTCH ligands, receptors, and target genes by HFFs incubated with recombinant human EREG (n=5). (G) HES1 expression fold change in SSc fibroblasts after incubation with EREG for 4 hours, n=5 per group. (H) EREG relative expression by BMDC primed with IFNα2 prior to exposure to NOTCH ligand DLL4 (n=3–4 per time point in each group). Statistics compare each group ± DLL4. (I) Relative expression of EGFR ligands by HFF (n=3). Genes with fewer than three points were below detectable level. (J) Changes in ECM gene expression when HFF were incubated with media alone (NT) or EREG neutralizing antibody (Ereg Ab). FNEDA refers to the extra domain A-containing isoform of fibronectin (n=5 per group). (K) Model of EREG-NOTCH circuit between monocyte-derived DC3 and fibroblasts. Data are means ± SD (ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P<0.0001) analyzed with unpaired two-tailed Student t test (A-H, J) and one-way ANOVA with Tukey multiple-comparisons test (I).

Article Snippet: EREG neutralizing antibody (R&D Systems MAB1068 clone 189611) or mouse IgG2a isotype control (Bio X Cell BE0085) 10 mg/kg diluted in 100 μl PBS was given subcutaneously twice weekly on the dorsal neck of anesthetized mice.

Techniques: Expressing, Incubation, Isolation, Cell Culture, Recombinant, Gene Expression, Derivative Assay, Two Tailed Test

Journal: Science immunology

Article Title: Epiregulin is a dendritic cell-derived EGFR ligand that maintains skin and lung fibrosis

doi: 10.1126/sciimmunol.abq6691

Figure Lengend Snippet: (A) Experimental diagram depicting adjacent punch biopsies obtained from the forearm of a patient with diffuse cutaneous SSc, which were cultured for 9 days in media alone (NT) or with addition of EREG neutralizing antibody (Ereg Ab). (B) Histology of cultured skin explants, with inset showing higher magnification of dermal collagen. (C, D) Skin explant media was analyzed for pro-COL1A1 N-terminal peptide (PINP) and TNC. (E) Percent reduction of protein by Ereg Ab treatment compared to NT control. (F) LDH activity of skin explant supernatants from patient SSc7. (G-N) Fresh explanted lung tissue from a deceased patient donor with familial idiopathic pulmonary fibrosis was processed for histologic staining, which showed fibroblastic foci formation and hyperplasia of alveolar type II epithelial cells, indicated by arrows (left panel H&E, right panel trichrome). The same tissue was cut into cubes and cultured for 10 days in the presence of the multikinase inhibitor nintedanib (Nin), the Alk5 inhibitor A-1544033 (IN-1130) (Alk5i), EREG antibody (Ereg Ab) or non-treated vehicle control (NT). (H-K) Relative expression of indicated genes, n=4 per group. (L-N) Protein secretion of indicated genes measured by ELISA, n=8 per group. ELISA samples with poor signal and qPCR outliers identified by Grubbs’s test with alpha = 0.05 were excluded. Data are means ± SD (ns, not significant, *P < 0.05, **P < 0.01, ****P < 0.0001) analyzed with paired two-tailed Student t test (C-E) comparing NT and Ereg Ab treated samples. In (H-N), comparison of each inhibitor to NT control was analyzed by one-way ANOVA with Dunnett’s multiple-comparisons test whereas Ereg Ab was individually compared to Nin and Alk5i by unpaired two-tailed Student t test.

Article Snippet: EREG neutralizing antibody (R&D Systems MAB1068 clone 189611) or mouse IgG2a isotype control (Bio X Cell BE0085) 10 mg/kg diluted in 100 μl PBS was given subcutaneously twice weekly on the dorsal neck of anesthetized mice.

Techniques: Cell Culture, Control, Activity Assay, Staining, Expressing, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Comparison

Journal: Science immunology

Article Title: Epiregulin is a dendritic cell-derived EGFR ligand that maintains skin and lung fibrosis

doi: 10.1126/sciimmunol.abq6691

Figure Lengend Snippet: (A-C) B6 mice were injected subcutaneously with 0.2 mg bleomycin (BLM) and 3 weeks later skin was stained for hematoxylin and eosin (A) and trichrome (B). Epidermis (epi), dermis (dermis) and dermal white adipose tissue (DWAT) are highlighted on histology. (C, D) Immunofluorescence images of PBS and BLM-treated skin 3 weeks post-injection. (E) Hydroxyproline content of the skin at different time points after subcutaneous bleomycin injection (n=3 per group). (F) Heatmap of mean log2(expression fold change) of ECM genes and EGFR ligands at different time points after subcutaneous bleomycin injection compared to the mean of each group and PBS controls, n=3 per time point. (G) Bulk RNA sequencing of dendritic cells isolated from fibrotic skin of Mgl2DTReGFPpANeo mice 3 weeks after subcutaneous bleomycin injection compared to PBS controls (n=3 per group). (H) Relative expression of Ereg at different time points after intratracheal bleomycin administration to B6 mice. (I) B6 mice were injected with bleomycin subcutaneously, then at 2 weeks injected intraperitoneally with Ifnar1-blocking antibody (Ifnar1 Ab), isotype control antibody (iso) or not treated (NT). No significant differences were found between NT and isotype Ab control groups, so they were combined for clarity. At 3 weeks, skin was analyzed for histology (J), dermal skin thickness (K), hydroxyproline (L), and gene expression (M, N). Data from E and F, G, and H are single independent experiments. Data in J-N are aggregated from two separate experiments. Data are means ± SD (ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P<0.0001) analyzed with one-way ANOVA with Tukey multiple-comparisons test (E, F, H, K) and unpaired two-tailed Student t test (L-N).

Article Snippet: EREG neutralizing antibody (R&D Systems MAB1068 clone 189611) or mouse IgG2a isotype control (Bio X Cell BE0085) 10 mg/kg diluted in 100 μl PBS was given subcutaneously twice weekly on the dorsal neck of anesthetized mice.

Techniques: Injection, Staining, Immunofluorescence, Expressing, RNA Sequencing, Isolation, Blocking Assay, Control, Gene Expression, Two Tailed Test

Journal: Science immunology

Article Title: Epiregulin is a dendritic cell-derived EGFR ligand that maintains skin and lung fibrosis

doi: 10.1126/sciimmunol.abq6691

Figure Lengend Snippet: (A-C) Diagramed in (A), cohorts of B6 and Ereg−/− mice were injected with bleomycin subcutaneously and 35 days later analyzed for skin thickness (B) and histology (C). (D-I) Diagramed in (D), 21 days after bleomycin injection mice began treatment with Ereg antibody (Ereg Ab) compared to controls treated with PBS (NT) for two weeks. Skin was analyzed for dermal thickness (E), hydroxyproline (F), gene expression (G, H) and histology (I), with H&E staining on the top row, trichrome in the middle row, and pEGFR immunohistochemistry (IHC) on the bottom row, n=8 (PBS), 11 (BLM), and 12 (Ereg Ab). (J) B6 and Ereg−/− mice were injected with bleomycin or PBS and 3 weeks later B6 mice were treated for 1 week with Ereg Ab or isotype control Ab, n=3 (PBS), 5 (isotype Ab), 5 (Ereg Ab), and 5 NT Ereg−/−. (K-L) As diagramed in (K), 10 days after intratracheal bleomycin mice were treated with Ereg Ab for two weeks. Lungs were analyzed for histology (L), modified Ashcroft score (M), hydroxyproline (N) and Ereg gene expression (O), n=6 (PBS), 11 (BLM), and 7 (Ereg Ab). Histology images of skin and lung are 10x and 20x magnification, respectively. IHC images are 40x magnification. Data are means ± SD (ns, not significant, *P < 0.05, **P < 0.01, ***P<0.001, ****P<0.0001) analyzed with unpaired two-tailed Student t test (F-H, N, O) and one-way ANOVA with Tukey multiple-comparisons test (B, E, J, M). Data for J is single experiment and data for A-C, D-I, and K-O are aggregated from two independent experiments.

Article Snippet: EREG neutralizing antibody (R&D Systems MAB1068 clone 189611) or mouse IgG2a isotype control (Bio X Cell BE0085) 10 mg/kg diluted in 100 μl PBS was given subcutaneously twice weekly on the dorsal neck of anesthetized mice.

Techniques: Injection, Gene Expression, Staining, Immunohistochemistry, Control, Modification, Two Tailed Test

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