Journal: Journal of Biological Chemistry

Article Title: Activation of Epidermal Growth Factor Receptor in Macrophages Mediates Feedback Inhibition of M2 Polarization and Gastrointestinal Tumor Cell Growth

doi: 10.1074/jbc.m116.750182

Figure Lengend Snippet: FIGURE 2. IL-4 stimulates HB-EGF production in macrophages, which is required for EGFR transactivation. A, Raw 264.7 cells were treated with IL-4 (10 ng/ml) for the indicated time periods. Levels of HB-EGF in cell culture supernatants were determined using ELISA. The HB-EGF level was expressed aspg/mlmedium.*,p0.05comparedwithuntreatedRaw264.7cells.B,raw 264.7 cells were treated with IL-4 (10 ng/ml) for the indicated time periods in the presence and absence of a 1-h pretreatment of the broad-spectrum MMP inhibitors GM6001 (2.5 M) and TAPI-1 (10 M). C, raw 264.7 cells transduced with siRNA against HB-EGF or non-targeting siRNA were treated with IL-4 (10 ng/ml) for 0.5 h. Cellular lysates were collected for Western blot analysis of EGFR expression and phosphorylation on tyrosine 1068 (EGFR-Tyr-1068) and HB-EGF expression. The -actin blot was used as a protein loading control. Data are quantified from at least three independent experiments in A and are representative of at least three independent experiments in B and C.

Article Snippet: Cell culture media were collected for determining the levels of HB-EGF using mouse HB-EGF ELISA kits (DuoSet ELISA Development System, R&D Systems, Inc.), according to the manufacturer’s instructions.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Transduction, Western Blot, Expressing, Phospho-proteomics, Control

Journal: Journal of Biological Chemistry

Article Title: Activation of Epidermal Growth Factor Receptor in Macrophages Mediates Feedback Inhibition of M2 Polarization and Gastrointestinal Tumor Cell Growth

doi: 10.1074/jbc.m116.750182

Figure Lengend Snippet: FIGURE 5. Transactivation of EGFR inhibits IL-4-stimulated HB-EGF gene expression and production in macrophages. Raw 264.7 mouse macro- phages and peritoneal macrophages isolated from indicated mice were treated with IL-4 (10 ng/ml) for 1 h (A and B), the indicated time periods (C), and 24 h (D and E). Levels of HB-EGF in cell culture supernatants were deter- mined using ELISA (A and B). The HB-EGF level was expressed as pg/ml medium. The levels of HB-EGF gene expression were quantified using real- timePCRanalysis(C–E).TheexpressionlevelsinuntreatedRaw264.7cellsand untreated WT and Egfrfl/fl peritoneal macrophages were set as 100% for com- parison with other groups. *, p 0.05 compared with untreated Raw 264.7 cells and WT and Egfrfl/fl macrophages. #, p 0.05 compared with the IL-4- treated WT and Egfrfl/fl macrophages.

Article Snippet: Cell culture media were collected for determining the levels of HB-EGF using mouse HB-EGF ELISA kits (DuoSet ELISA Development System, R&D Systems, Inc.), according to the manufacturer’s instructions.

Techniques: Gene Expression, Isolation, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Journal of Biological Chemistry

Article Title: Activation of Epidermal Growth Factor Receptor in Macrophages Mediates Feedback Inhibition of M2 Polarization and Gastrointestinal Tumor Cell Growth

doi: 10.1074/jbc.m116.750182

Figure Lengend Snippet: Figure 3. LPS stimulates HB-EGF production in macrophages, which is not affected by inhibition of EGFR kinase activity. Raw 264.7 cells were treated with LPS (1 µg/ml) in the presence and absence of 1- h pretreatment of an EGFR tyrosine kinase inhibitor, AG1478 (450 nM) for the indicated time periods. Levels of HB-EGF in cell culture supernatants were examined using ELISA assay. Cells were lysed and the protein concentrations were detected. The HB- EGF level was expressed as pg/mg soluble proteins. * p<0.05 compared to untreated Raw 264.7 cells.

Article Snippet: Cell culture media were collected for determining the levels of HB-EGF using mouse HB-EGF ELISA kits (DuoSet ELISA Development System, R&D Systems, Inc.), according to the manufacturer’s instructions.

Techniques: Inhibition, Activity Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Life Science Alliance

Article Title: The ADAM17 sheddase complex regulator iTAP/Frmd8 modulates inflammation and tumor growth

doi: 10.26508/lsa.202201644

Figure Lengend Snippet: (A, C) Immunoblots showing expression levels of immature versus mature ADAM17 (A17) in parental (PAR.) versus iTAP/Frmd8 KO LLC cells (A) or empty vector (EV)–transduced versus iTAP/Frmd8-overexpressing (OE) LLC cells (C). Protein samples were deglycosylated with Endo-H (H) and PNGase F (F) versus mock (M). In (C), the left-hand immunoblot shows expression levels of Flag-tagged iTAP in EV-transduced versus iTAP/Frmd8-overexpressing (OE) LLC cells. Immunoblots for the transferrin receptor serve as a loading control. Immature ADAM17 is indicated with white arrowheads; the black arrowhead denotes both glycosylated mature ADAM17 and deglycosylated immature ADAM17, respectively (which have similar electrophoretic mobility), whereas red arrowheads denote the fully deglycosylated, mature, ADAM17 polypeptide. The asterisk indicates an unspecific band. (B, D) Relative mRNA expression levels of iTAP/Frmd8 in parental versus iTAP/Frmd8 KO LLC cells (B) and EV-transduced control cells versus iTAP/Frmd8-overexpressing (OE) LLC cells (D). Expression was normalized to the levels of a housekeeping control mRNA ( Gapdh ). (E, F) Soluble amphiregulin levels in the culture medium of parental versus iTAP/Frmd8 KO cells (E) or EV control cells versus iTAP/Frmd8-overexpressing (OE) LLC cells (F). The cells were preincubated with DMSO, the metalloprotease inhibitor MM (5 μM), or the ADAM10-specific inhibitor GI254023X (GI) (1 μM) and then treated with DMSO or PMA (1 μM). n = 3. (G) Schematic illustrating the protocol for the subcutaneous (SC) tumor model. (H) Volume of subcutaneous tumors in WT immunocompetent mice receiving iTAP/Frmd8 KO LLC cells or iTAP/Frmd8-overexpressing (OE) LLC cells over time compared with their respective controls. (I, J) Tumor volume (I) and tumor weight (J) of iTAP/Frmd8 KO LLC cells or iTAP/Frmd8-overexpressing (OE) LLC cells compared with their respective controls injected into WT mice. (K, L) Proportion of Ki67-positive nuclei relative to controls (K) and representative images (L) of the tumors described above. n = 3 with three to six mice per genotype per experiment. For Ki67 analysis, n = 2 with two to three mice per genotype. Results are indicated as the mean ± SEM. A scale bar is indicated within the Ki67 IF images of 78 μm. The Mann–Whitney–Wilcoxon test was used; * represents P < 0.05, ** represents P < 0.01, and **** represents P < 0.0001.

Article Snippet: Then, the cells were stimulated with PMA at 1 μM (P1585; Sigma-Aldrich) or DMSO for 1 h. The medium was collected and, after clearance, was used to evaluate the levels of TNF, HB-EGF, and amphiregulin using specific ELISA kits (88-7324-22, eBioscience; DY8239-05, R&D Systems; and DY989, R&D Systems, respectively).

Techniques: Western Blot, Expressing, Plasmid Preparation, Control, Injection, MANN-WHITNEY

Journal: Life Science Alliance

Article Title: HB-EGF activates EGFR to induce reactive neural stem cells in the mouse hippocampus after seizures

doi: 10.26508/lsa.202201840

Figure Lengend Snippet: HB-EGF increases early after the induction of MTLE in vivo and is secreted by NSPCs in vitro. (A) Confocal microscopy images from Nestin-GFP mice, 14dpKA showing the reduction caused by gefitinib of the MTLE-induced GCD and NSC overactivation. Scale bar, 20 μm. (B) Quantification of the volume of the GCL in saline+DMSO–, saline+ gefitinib–, KA+DMSO–, and KA+gefitinib–treated animals 14dpKA. (C) Quantification of the BrdU+ cells in saline+DMSO–, saline+ gefitinib–, KA+DMSO–, and KA+gefitinib–treated animals 14dpKA. (D) Quantification of BrdU+ NSCs, ANPs, and overall cells in the SGZ expressed as the percentage with respect to KA+DMSO animals. (E) Confocal microscopy images showing an increase in the EGFR ligand HB-EGF in the hilus and the molecular layer of Nestin-GFP mice 3dpKA. Scale bar, 20 μm. (F) Quantification of HB-EGF by ELISA at different time points during the first 3dpKA. Values are represented as the pg of HB-EGF in 100 μg of the tissue. (G) Immunofluorescence images of NSPCs in vitro showing HB-EGF expression. Scale bar, 10 μm. (H) Quantification by ELISA of HB-EGF released in vitro, expressed as the pg of the ligand per mL. Dots show individual data. Data information: for (B), Kruskal–Wallis test, * P < 0.05, * P < 0.01, and *** P < 0.001. Bars show the mean ± SEM. Dots show individual data. For (C), Kruskal–Wallis test, * P < 0.05, * P < 0.01, and *** P < 0.001. Bars show the mean ± SEM. Dots show individual data. For (D), t test, ** P < 0.01. Bars show the mean ± SEM. Dots show individual data. For (F), one-way ANOVA, * P < 0.05 and *** P < 0.001. Bars show the mean ± SEM. Dots show individual data. For (H), Kruskal–Wallis test, * P < 0.05. Bars show the mean ± SEM. Source data are available for this figure.

Article Snippet: Next, we determined the amount of HB-EGF per cell through ELISA (Cat#DY8239-05; R&D Systems).

Techniques: In Vivo, In Vitro, Confocal Microscopy, Saline, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Expressing