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: bioRxiv

Article Title: ERBB4-mediated signaling is a mediator of resistance to BTK and PI3K inhibitors in B cell lymphoid neoplasms

doi: 10.1101/2023.01.01.522017

Figure Lengend Snippet: (A) Expression of surface ERBB4 by FACS in parental (grey) and resistant (red) lines. Dashed black line for negative control (neg-cnt). Data derived from two independent experiments. (B) Secretion of HBEGF in parental (grey) and resistant (red) by ELISA. Data derived from three independent experiments. (C) Expression levels of surface CD19 (x-axis, PE-conjugated) and intracellular HBEGF (y-axis, APC-conjugated) by FACS in parental (left) and resistant (right) cells, percentage correspond to percentages of total viable cells. Data representative of two independent experiments. (D) Levels of protein phosphorylation by immunoblot in parental (grey) and resistant (red) cells. Values correspond to average of protein quantification normalized to GAPDH levels in three independent experiments. Error bars represent standard error of the mean. (E) Quantification of AKT phosphorylation by immunofluorescence, error bars represent standard error of the mean. Data derived from at least three independent experiments. P values from t-test, statistically significant for p< 0.05. (E) Detail of p-AKT expression by immunofluorescence in parental (PAR, top) and resistant (RES, bottom) upon DMSO (control, left) or 1μM idelalisib (right). (G) Cell viability by MTT assay of parental cells (PAR, black line) exposed to idelalisib in presence or not of conditioned medium from Karpas1718 idelalisib-resistant (RES, red line), cultured for 48h, PAR + RES-cond RPMI, dashed black line) or stimulated with 30ng/ml of recombinant HBEGF (dashed red line). Data derived from the average of three independent experiments. P values from a moderated t-test, statistically significant for p< 0.05.

Article Snippet: ELISA (Human HB-EGF Quantikine ELISA Kit, R&D Systems) was performed according to the manufacturer’s protocols.

Techniques: Expressing, Negative Control, Derivative Assay, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Western Blot, Immunofluorescence, Control, MTT Assay, Cell Culture, Recombinant

Journal: bioRxiv

Article Title: ERBB4-mediated signaling is a mediator of resistance to BTK and PI3K inhibitors in B cell lymphoid neoplasms

doi: 10.1101/2023.01.01.522017

Figure Lengend Snippet: (A) Small interfering RNAs were used for gene expression silencing of ERBB2 alone (yellow dashed line), ERBB4 alone (red dashed line) or concomitant silencing of ERBB2 and ERBB4 (brown dashed line). Black lines for resistant control. (B) miRNAs mimics were used for miR-29c expression (yellow dashed line), let-7c (red dashed line) or miR-30c (brown dashed line). Black lines for resistant control. Drug sensitivity was evaluated by MTT assay in resistant Karpas1718 cells for the PI3K inhibitors idelalisib, umbralisib, copanlisib and duvelisib. Data correspond of two independent experiments, error bars represent standard deviation of the mean. P values from a moderated t-test, statistically significant for p< 0.05. (C) Levels of HBEGF secretion by ELISA in resistant cells upon miRNA mimics for miR-29c (yellow), let-7c (red) or control (grey). Data derived from three independent experiments. P values from a moderated t-test, statistically significant for p< 0.05.

Article Snippet: ELISA (Human HB-EGF Quantikine ELISA Kit, R&D Systems) was performed according to the manufacturer’s protocols.

Techniques: Gene Expression, Control, Expressing, MTT Assay, Standard Deviation, Enzyme-linked Immunosorbent Assay, Derivative Assay

Journal: bioRxiv

Article Title: ERBB4-mediated signaling is a mediator of resistance to BTK and PI3K inhibitors in B cell lymphoid neoplasms

doi: 10.1101/2023.01.01.522017

Figure Lengend Snippet: (A) HBEGF secretion was evaluated in the serum of CLL samples at the end of the treatment by ELISA (Luminex, R&D Systems). (A) Patients responding (responders, blue) or non-responding (non-responders, red) to idelalisib were compared by t-test. (B) HBEGF secretion in the serum of patients treated with ibrutinib was compared at the time of progression (red) with baseline levels previously of ibrutinib treatment (pre-treatment, blue). P for adjusted p-value from a t-test. MFI: mean fluorescence intensity.

Article Snippet: ELISA (Human HB-EGF Quantikine ELISA Kit, R&D Systems) was performed according to the manufacturer’s protocols.

Techniques: Enzyme-linked Immunosorbent Assay, Luminex, Fluorescence

Journal: Microorganisms

Article Title: Effects of Spore-Displayed p75 Protein from Lacticaseibacillus rhamnosus GG on the Transcriptional Response of HT-29 Cells

doi: 10.3390/microorganisms10071276

Figure Lengend Snippet: Effect of CotG-p75 on HBEGF mRNA expression ( a ) and HBEGF protein production ( b ). In panel ( a ), cells were treated with various concentrations of CotG-p75 (10 5 to 10 7 spores/mL) for 3 h. In panel ( b ), cells were treated with CotG-p75 (10 7 spores/mL) during various times (0, 3, 6, and 12 h). The mRNA expression levels and protein production levels in treated groups were compared with the control group. All data are expressed as mean ± standard deviation ( n = 3). Asterisks (*) indicate a significance difference from the control (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: The level of HBEGF and MUC5AC protein was measured using an HBEGF ELISA kit (Assay Genie, Dublin, Ireland) and MUC5AC ELISA assay kit (Cusabio Biotech, Houston, TX, USA) following the manufacturer’s protocol.

Techniques: Expressing, Control, Standard Deviation