Journal: British Journal of Cancer

Article Title: Serum levels of hepatocyte growth factor and epiregulin are associated with the prognosis on anti-EGFR antibody treatment in KRAS wild-type metastatic colorectal cancer

doi: 10.1038/bjc.2014.230

Figure Lengend Snippet: Graphical representations of serum concentrations of ligands measured by ELISA . Individual data of serum EREG, HGF, EGF, AREG, NRG, IGF-1 and TGF- α are summarized by graphs. Blue bars show serum levels at pre-treatment and red bars show those at progression disease.

Article Snippet: Concentrations of EREG in serum were measured using Human epiregulin ELISA kit (CSB-EL007779HU, CUSABIO, Wuhan, China).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: British Journal of Cancer

Article Title: Serum levels of hepatocyte growth factor and epiregulin are associated with the prognosis on anti-EGFR antibody treatment in KRAS wild-type metastatic colorectal cancer

doi: 10.1038/bjc.2014.230

Figure Lengend Snippet: Survival curves of pretreatment hepatocyte growth factor (HGF) and epiregulin (EREG) levels among KRAS wild-type patients and all wild-type of KRAS , BRAF , PIK3CA and NRAS. Survival curves of PFS in terms of HGF levels are shown among ( A ) KRAS wild-type patients and ( B ) among all wild-type patients. Survival curves of PFS in terms of EREG levels are shown among ( C ) KRAS wild-type patients and ( D ) among all wild-type patients. Among KRAS wild-type patients, survival curves of OS in ( E ) HGF levels and ( F ) EREG levels are shown. Patients with low levels of ligands had longer OS compared with patients with high levels.

Article Snippet: Concentrations of EREG in serum were measured using Human epiregulin ELISA kit (CSB-EL007779HU, CUSABIO, Wuhan, China).

Techniques:

Journal: British Journal of Cancer

Article Title: Serum levels of hepatocyte growth factor and epiregulin are associated with the prognosis on anti-EGFR antibody treatment in KRAS wild-type metastatic colorectal cancer

doi: 10.1038/bjc.2014.230

Figure Lengend Snippet: Results of serum concentration of hepatocyte growth factor (HGF) and epiregulin (EREG) and genomic mutations of KRAS , BRAF , PIK3CA and NRAS

Article Snippet: Concentrations of EREG in serum were measured using Human epiregulin ELISA kit (CSB-EL007779HU, CUSABIO, Wuhan, China).

Techniques: Concentration Assay

Journal: British Journal of Cancer

Article Title: Serum levels of hepatocyte growth factor and epiregulin are associated with the prognosis on anti-EGFR antibody treatment in KRAS wild-type metastatic colorectal cancer

doi: 10.1038/bjc.2014.230

Figure Lengend Snippet: Objective response rate (ORR) and disease control rate (DCR) by pretreatment serum levels of hepatocyte growth factor (HGF) and epiregulin (EREG) when the cutoff values are median of serum concentration

Article Snippet: Concentrations of EREG in serum were measured using Human epiregulin ELISA kit (CSB-EL007779HU, CUSABIO, Wuhan, China).

Techniques: Control, Concentration Assay

Journal: British Journal of Cancer

Article Title: Serum levels of hepatocyte growth factor and epiregulin are associated with the prognosis on anti-EGFR antibody treatment in KRAS wild-type metastatic colorectal cancer

doi: 10.1038/bjc.2014.230

Figure Lengend Snippet: Survival curves by the change of serum levels of ligands after treatment. Survival curves of PFS by pretreatment levels of HGF and EREG (high/low) and change of serum levels at PD compared with pretreatment (elevation/no elevation) levels are shown. ( A ) PFS curves divided by change of serum HGF levels. ( B ) PFS curves divided by change of serum EREG levels. ( C ) OS curves divided by change of serum HGF levels. ( D ) OS curves divided by change of serum EREG levels.

Article Snippet: Concentrations of EREG in serum were measured using Human epiregulin ELISA kit (CSB-EL007779HU, CUSABIO, Wuhan, China).

Techniques:

Journal: British Journal of Cancer

Article Title: Serum levels of hepatocyte growth factor and epiregulin are associated with the prognosis on anti-EGFR antibody treatment in KRAS wild-type metastatic colorectal cancer

doi: 10.1038/bjc.2014.230

Figure Lengend Snippet: Objective response rate (ORR) and disease control rate (DCR) by pretreatment serum levels of hepatocyte growth factor (HGF) and epiregulin (EREG) when the appropriate cutoff values were evaluated by ROC curve analysis

Article Snippet: Concentrations of EREG in serum were measured using Human epiregulin ELISA kit (CSB-EL007779HU, CUSABIO, Wuhan, China).

Techniques: Control

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: 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: Vaginas were collected for immunofluorescence of Raptor and Phospho-S6 in Fig. . For examining the effect of EREG, an intravaginal injection of 400 ng EREG protein (50599-M01H, Sinobiological) or PBS was given to OVX mice for 3 days, and the mice were sacrificed 24 h after the last injection.

Techniques: Expressing, Immunofluorescence, 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: 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-ER-alpha 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, Immunofluorescence, 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: A mTORC1 signaling participates in the proliferation and differentiation of vaginal epithelium by promoting the expression level of PR and EREG-YAP1 in Rptor fl/fl mice. B Loss of Rptor compromises the estrogen-induced proliferation and differentiation 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-ER-alpha 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: Frontiers in Oncology

Article Title: Epiregulin expression and secretion is increased in castration-resistant prostate cancer

doi: 10.3389/fonc.2023.1107021

Figure Lengend Snippet: EREG expression and secretion is higher in CRPC in comparison to LNCaP cells (CSPC). (A) The expression of EREG after ADT treatment was assumed to be increased according to preliminary data. Analysis of EREG expression using qRT-PCR show an increased (p < 0.0001) EREG expression in CRPC cells in comparison to castration sensitive LNCaP cells. (B) Flow cytometry analysis shows the presence of EREG (dark) on the cell surface of CSPC and CRPC cell lines. Goat-IgG (light) is used as a control. Histograms show representative flow cytometry results for CSPC and CRPC cell lines using EREG antibody and goat-IgG. (C) The ratio is determined from the mean of the fluorescence intensity of the sample compared to the control. EREG protein on cell surface of 22Rv1 (p=0.0043) and LNCaP EnzR (p < 0.0001) cells is significantly increased in comparison to CSPC cells whereas EREG protein on DU145 (p=0.55) and PC3 (p=0.29) is slightly, but not significant increased compared to CSPC cells. (D) PCa cell culture supernatant was collected and EREG protein was investigated by sandwich ELISA. Whereas LNCaP and 22Rv1 cells secrete no EREG protein, it was detected in DU145 (330 pg/ml), PC3 (66 pg/ml) and LNCaP EnzR (37 pg/ml) supernatant. All graphs show the mean and SD from four independently performed experiments (ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

Article Snippet: The protein content of EREG in the cell culture supernatant was determined by sandwich ELISA using the Human Epiregulin DuoSet ELISA (R&D Systems, Minneapolis, Minnesota, US) according to the manufacturer’s instructions.

Techniques: Expressing, Comparison, Quantitative RT-PCR, Flow Cytometry, Control, Fluorescence, Cell Culture, Sandwich ELISA

Journal: Frontiers in Oncology

Article Title: Epiregulin expression and secretion is increased in castration-resistant prostate cancer

doi: 10.3389/fonc.2023.1107021

Figure Lengend Snippet: Amount of EREG correlates with increasing aggressiveness of PCa samples. TMA with 196 samples were immunohistochemically stained with EREG antibody. (A) Representative overview and detail pictures of EREG staining in PCa samples for different categories of IRS (Overview pictures: Magnification 200x, scale bar 200 µm; Detail pictures in black boxes: Magnification 630x, scale bar 100 µm). Samples with an IRS of 0 exhibit no expression of EREG, while samples with an IRS of 1 show a weak EREG expression. An IRS of 2 indicates an intermediate EREG expression and an IRS of 3 a strong expression. (B) The pie chart shows the distribution of cases split by IRS for EREG. (C) Violin plot with correlation of EREG IRS and preoperative PSA levels (median: dashed line, 95% quartile: dotted line). The EREG IRS tends to correlate positively with PSA level without significance (p=0.29; ns, not significant). (D–H) Bubble plots show the distribution of EREG IRS in relation to (D) PCa related death, (E) PCa grading, (F) tumor recurrence, (G) CHGA IRS and (H) SYP IRS. The size of the bubbles indicates the relative number of cases, while the numbers to the right of the bubbles represent the absolute number of cases. CHGA and SYP IRS were determined in the same manner as for EREG.

Article Snippet: The protein content of EREG in the cell culture supernatant was determined by sandwich ELISA using the Human Epiregulin DuoSet ELISA (R&D Systems, Minneapolis, Minnesota, US) according to the manufacturer’s instructions.

Techniques: Staining, Expressing

Journal: Frontiers in Oncology

Article Title: Epiregulin expression and secretion is increased in castration-resistant prostate cancer

doi: 10.3389/fonc.2023.1107021

Figure Lengend Snippet: Elevated phosphorylation of SMAD2/3 seems to enhance EREG expression in PCa cells. (A) Column graph shows ERK1/2 phosphorylation at Thr202/Tyr204 in PCa cell lines LNCaP, 22Rv1, DU145 and PC3. ERK1/2 phosphorylation in 22Rv1 and DU145 cells are increased in comparison to LNCaP cells. Additionally, a representative immunoblot image for ERK1/2 phosphorylation in PCa cell lines is illustrated. (B) ERK1/2 phosphorylation at Thr202/Tyr204 of LNCaP in comparison to LNCaP EnzR cells and a representative immunoblot image are shown. Phosphorylation of ERK1/2 is enhanced in LNCaP EnzR cells. (C) The column graph shows the phosphorylation of transcription factor ETS1 at Thr38 in the different PCa cell lines without differences in ETS1 phosphorylation and expression. The adjacent representative immunoblot illustrates the result. (D) As revealed in the graph and the representative immunoblot, LNCaP EnzR exhibit no alteration in ETS1 phosphorylation at Thr38 compared with the parental LNCaP cells as well. (E) The bar graph and representative immunoblots shows the phosphorylation of transcription factor SMAD2/3 at Ser465, Ser467, Ser423, Ser425 in PCa cell lines. Compared to LNCaP cells, SMAD2/3 phosphorylation in 22Rv1 and DU145 is slightly increased, but PC3 cells show a noticeably higher phosphorylation of SMAD2/3. (F) SMAD2/3 phosphorylation of LNCaP EnzR cells is illustrated in a column graph and a representative immunoblot image. Phosphorylation in SMAD2/3 at Ser465, Ser467, Ser423, Ser425 is increased in LNCaP EnzR in comparison to parental LNCaP cells. All graphs show the mean and SD from four independently performed experiments. (G) Total EREG protein in cell culture supernatant of DU145 cells after TGF-β1 (p=0.2649) or TGF-β2 (p=0.0283) treatment is significantly elevated in comparison to untreated DU145 cells. All graphs show the mean and SD from four independent experiments (ns, not significant; *p < 0.05).

Article Snippet: The protein content of EREG in the cell culture supernatant was determined by sandwich ELISA using the Human Epiregulin DuoSet ELISA (R&D Systems, Minneapolis, Minnesota, US) according to the manufacturer’s instructions.

Techniques: Phospho-proteomics, Expressing, Comparison, Western Blot, Cell Culture

Journal: Frontiers in Oncology

Article Title: Epiregulin expression and secretion is increased in castration-resistant prostate cancer

doi: 10.3389/fonc.2023.1107021

Figure Lengend Snippet: EREG protein biosynthesis is post-transcriptionally regulated by miRNAs. (A) The expression of miR-19a and -19b that were assumed to be reduced according to preliminary data was assessed by qRT-PCR. MiR-19a (p=0.0001) and -19b (p=0.0053) were significantly reduced in LNCaP EnzR compared to parental LNCaP cells. (B) Predicted miRNA binding sites in the 3’UTR of EREG mRNA for miR-19a, -19b, -20b and the mutated miRNA binding sites are shown. The bold nucleotides in the sequences represent the miRNAs seed sequence and its corresponding binding sites. (C–E) For luciferase reporter assay, miRNA expression plasmids (control grey bar, miRNA black bar) were cotransfected with empty reporter plasmid (control), reporter gene construct containing wildtype EREG 3’UTR (WT) or reporter gene construct containing mutated EREG 3’UTR (MUT). The luciferase activity of the reporter gene plasmid coexpressed with control expression plasmid was set to 1. MiR-19a (p=0.0001), -19b (p=0.0006) and -20b (p<0.0001) significantly reduce luciferase activity of the reporter gene construct containing EREG 3’UTR in comparison to the control. After mutation of the seed sequence, the corresponding miRNAs are not able to reduce the luciferase activity. (F) Flow cytometry analysis using EREG antibody show the EREG presence on the cell surface of LNCaP EnzR cells transfected with miRNA expression plasmid. The histograms show representative flow cytometry analyses of LNCaP EnzR cells transfected with miRNA expression plasmid using EREG antibody (dark) and goat-IgG (light). (G) The ratio is determined from the mean of the fluorescence intensity of the specific EREG antibody compared to the control (goat-IgG). EREG protein on cell surface of LNCaP EnzR cells transfected with expression plasmids for miR-19a (p=0.0008), miR-19b (p=0.001) or miR-20b (p=0.0015) is significantly decreased in comparison to LNCaP EnzR cells transfected with control expression plasmid. All graphs show the mean and SD from four independent experiments (ns not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). (H) Total EREG protein in cell culture supernatant of DU145 cells transfected with expression plasmids for miR-19a (p=0.0015), miR-19b (p=0.0029) or miR-20b (p=0.038) is significantly decreased in comparison to DU145 cells transfected with control expression plasmid. All graphs show the mean and SD from four independent experiments (ns, not significant; *p < 0.05; **p < 0.01).

Article Snippet: The protein content of EREG in the cell culture supernatant was determined by sandwich ELISA using the Human Epiregulin DuoSet ELISA (R&D Systems, Minneapolis, Minnesota, US) according to the manufacturer’s instructions.

Techniques: Expressing, Quantitative RT-PCR, Binding Assay, Sequencing, Luciferase, Reporter Assay, Control, Plasmid Preparation, Construct, Activity Assay, Comparison, Mutagenesis, Flow Cytometry, Transfection, Fluorescence, Cell Culture

Journal: Frontiers in Oncology

Article Title: Epiregulin expression and secretion is increased in castration-resistant prostate cancer

doi: 10.3389/fonc.2023.1107021

Figure Lengend Snippet: Proteases, shedding the ectodomain of EREG are increased in CRPC compared to CSPC cells. (A) The dot plot depicts the ADAM17 expression of CSPC and CRPC cell lines using qRT-PCR analysis. 22Rv1, DU145, PC3 and LNCaP EnzR (p<0.0001) significantly express more ADAM17 compared to LNCaP cells. (B) Flow cytometry analysis using ADAM17 antibody show the ADAM17 presence on the cell surface of CSPC and CRPC cell lines. ADAM17 protein level on cell surface of PC3 (p<0.0001) and LNCaP EnzR (p < 0.0001) cells is increased in comparison to LNCaP cells whereas ADAM17 protein content on 22Rv1 and DU145 is not altered. (C) The pictures show representative flow cytometry analysis of CSPC and CRPC cell lines using ADAM17 antibody (dark) and mouse-IgG (light) as a control. MMP2 (D) and MMP9 (E) expression in CSPC and CRPC cell lines was determined by qRT-PCR. In comparison to LNCaP cells 22Rv1 (p=0.0057), PC3 (p=0.0002) and LNCaP EnzR (p < 0.0001) cells show an increased expression of MMP2. The MMP9 expression of 22Rv1 (p < 0.0001), DU145 (p < 0.0001), PC3 (p < 0.0001) and LNCaP EnzR (p < 0.0001) is enhanced compared to LNCaP cells. (F) Solid phase ELISA using a specific MMP2 antibody demonstrates the secretion of MMP2 in cell culture supernatant of PCa cell lines. The ELISA results reveal that MMP2 is secreted by all five PCa cell lines, but 22Rv1 (p=0.0021), DU145 (p=0.0026), PC3 (p < 0.0001) and LNCaP EnzR (p < 0.0001) cells secrete a higher amount of MMP2 protein in comparison to LNCaP cells. (G) PCa cell culture supernatant was collected and MMP9 protein was investigated by solid phase ELISA. All PCa cells secrete MMP9 protein, but the CRPC cell lines 22Rv1 (p < 0.0001), DU145 (p=0.0005), PC3 (p < 0.0001) and LNCaP EnzR (p < 0.0001) secrete more MMP9 protein than CSPC cell line LNCaP. All graphs show the mean and SD from four independent experiments (ns, not significant; **p < 0.01; ***p < 0.001; ****p < 0.0001).

Article Snippet: The protein content of EREG in the cell culture supernatant was determined by sandwich ELISA using the Human Epiregulin DuoSet ELISA (R&D Systems, Minneapolis, Minnesota, US) according to the manufacturer’s instructions.

Techniques: Expressing, Quantitative RT-PCR, Flow Cytometry, Comparison, Control, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Biology of reproduction

Article Title: Effect of epidermal growth factor-like peptides on the metabolism of in vitro- matured mouse oocytes and cumulus cells.

doi: 10.1095/biolreprod.113.115311

Figure Lengend Snippet: FIG. 3. Effect of FSH, EGF, and EGF-like peptides on COC b-O-linked glycosylation. A1) Protein b-O-linked glycosylation was examined at 12 h IVM in the presence of control (no treatment), FSH, AREG, EREG, BTC, or EGF and b-O-linked glycosylation (CTD110.6) and nuclear staining (PI) fluorescence were imaged. Images shown are representative of 30 COCs per treatment group over three replicate experiments. Original magnification 360. A2) Quantification of relative CTD110.6 fluorescence in cumulus cells and oocytes. B) COC mRNA expression of Ogt was measured at 6 h IVM (n ¼ 6). Bars not sharing a common letter are significantly different (P , 0.02). The data represent means 6 SEM.

Article Snippet: COC IVM IVM COCs were cultured in bicarbonate buffered aMEM (Gibco) supplemented with 3 mg/ml BSA and either recombinant human FSH (50 mIU/ml; Puregon; Organon, Oss, The Netherlands), recombinant human EGF (10 ng/ml; R&D Systems, Minneapolis, MN), recombinant mouse AREG (50 ng/ml; R&D Systems), recombinant mouse EREG (50 ng/ml; R&D Systems), or recombinant mouse BTC (50 ng/ml; R&D Systems), at 378C with 5% CO 2 in air.

Techniques: Glycoproteomics, Control, Staining, Fluorescence, Expressing

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