Journal: Journal of Biological Chemistry

Article Title: Elastase-released Epidermal Growth Factor Recruits Epidermal Growth Factor Receptor and Extracellular Signal-regulated Kinases to Down-regulate Tropoelastin mRNA in Lung Fibroblasts

doi: 10.1074/jbc.m200243200

Figure Lengend Snippet: FIG. 5. NE-initiated ERK activation is not mediated by release of bFGF, PDGF, or TGF-. Serum-starved lung fibroblasts (in 35-mm plates) were incubated for 30 min with 100 g/ml control, anti-bFGF, anti-PDGF, or anti-TGF--neutralizing antibodies and then challenged for 15 min with 10 g/ml NE or 10 ng/ml bFGF, PDGF, TGF-, EGF, or hbEGF as indicated. Status of ERK activation and total ERK amounts were determined by Western blot with anti-phospho-ERK and anti- ERK antibodies, respectively.

Article Snippet: Goat polyclonal anti-human TGF- (neutralizing, catalog number AF-239-NA) and goat polyclonal anti-human hbEGF (neutralizing, catalog number AF-259-NA) antibodies, recombinant human EGF, hbEGF, TGF- , betacellulin, amphiregulin (long form), and heregulin 1 (EGF domain) were obtained from R & D Systems.

Techniques: Activation Assay, Incubation, Control, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Elastase-released Epidermal Growth Factor Recruits Epidermal Growth Factor Receptor and Extracellular Signal-regulated Kinases to Down-regulate Tropoelastin mRNA in Lung Fibroblasts

doi: 10.1074/jbc.m200243200

Figure Lengend Snippet: FIG. 9. NE initiates ERK activation by releasing EGF. A, serum-starved lung fibroblasts (in 100-mm plates) were incubated with or without 5 g/ml NE or 5 g/ml PE for the indicated times, and then conditioned medium samples, as well as total cell lysates, were harvested in the presence of 1 mM DFP. Conditioned media samples (initial volume 12 ml) were concentrated (final volume 500 l) and dialyzed against 10 mM Tris-HCl, pH 7.5, at 4 °C on microconcentrators (Amicon), and 50 l of each sample were analyzed by Western blot (in non-reducing conditions) with anti-EGF neutralizing antibodies (upper part). The electrophoretic mobility of protein standards (Bio-Rad) is shown on the right. ERK activation status and total ERK in total cell lysates were determined by Western blot (in non-reducing conditions) with anti-phospho-ERK and anti-ERK antibodies, respectively (bottom part). B, serum-starved lung fibroblasts (in 12-well cluster plates) were incubated for 30 min in the presence of 100 g/ml control or anti-EGF-neutralizing antibodies (left part) or with or without 10 M AG1478 (right part). Then cells were challenged for 15 min with 100 l of the concentrated and dialyzed conditioned medium samples isolated from the control or NE-treated cells (A), 5 g/ml NE, 10 ng/ml EGF, 10 ng/ml TGF-, 10 ng/ml hbEGF, or 10 ng/ml betacellulin as indicated. Status of ERK activation and total ERK were determined by Western blot with anti-phospho-ERK and anti-ERK antibodies, respectively.

Article Snippet: Goat polyclonal anti-human TGF- (neutralizing, catalog number AF-239-NA) and goat polyclonal anti-human hbEGF (neutralizing, catalog number AF-259-NA) antibodies, recombinant human EGF, hbEGF, TGF- , betacellulin, amphiregulin (long form), and heregulin 1 (EGF domain) were obtained from R & D Systems.

Techniques: Activation Assay, Incubation, Western Blot, Control, Isolation

Journal: Breast Cancer Research : BCR

Article Title: BHLHE40 confers a pro-survival and pro-metastatic phenotype to breast cancer cells by modulating HBEGF secretion

doi: 10.1186/s13058-018-1046-3

Figure Lengend Snippet: The HIF-BHLHE40-HBEGF axis plays a role in promoting cell migration during wound healing. a Monolayer scratch increased protein levels of HIF1A, BHLHE40, and HBEGF in MDA-MB-231 empty vector (EV) cells. Intensive scratch wounds were generated using EMD Millipore’s Cell Comb scratch assay kit and immunoblotting was performed 6 h after cells cultured under normoxia (19% O 2 ) or hypoxia (1% O 2 ). b BHLHE40-knockout (KO) by CRISPR/Cas9 editing reduced the migratory activity of MDA-MB-231 cells, which was restored by the addition of HBEGF peptide into the culture medium. In contrast, a HBEGF-neutralizing antibody reduced the migratory activity of MDA-MB-231-EV cells. Real-time assessment of migratory activity after wound scratch was performed using the IncuCyte ZOOM-ImageLock plate system. * p < 0.05 ( n = 6, time points 6–24 h, HBEGF vs. untreated), ** p < 0.05 ( n = 6, time points 6–24 h, anti-HBEGF vs. untreated), one-way ANOVA followed by Tukey’s post-hoc tests. Representative data from two independent experiments with six replicates are presented. c Images of representative wound fields at 0 and 24 h after wound scratch as described in b . d Conditioned medium or purified exosomes from MDA-MB-231-EV cells (24 h after wound scratch) increased migratory activities of MDA-MB-231 BHLHE40-KO cells, as determined by the transwell migration assays. The migrated cells in six fields were imaged and counted under fluorescent microscopy. The results are presented as: percent migration = mean number of cells migrating through the uncoated transwells × 100/mean number of seeded cells; * p < 0.05 ( n = 12, vs. untreated control), one-way ANOVA followed by Tukey’s post-hoc tests

Article Snippet: To examine the effect of HBEGF on cell migration and invasion, a neutralizing antibody to HBEGF (10 μg/ml; AF-259-NA, R&D systems, Minneapolis, MN, USA) or a HBEGF peptide (20 μg/ml) was added to the medium.

Techniques: Migration, Plasmid Preparation, Generated, Wound Healing Assay, Western Blot, Cell Culture, Knock-Out, CRISPR, Activity Assay, Purification, Microscopy, Control