Journal: Gastroenterology

Article Title: Toll-like receptor-4 promotes the development of colitis-associated colorectal tumors.

doi: 10.1053/j.gastro.2007.09.008

Figure Lengend Snippet: Figure 5. TLR4 signal regulates EGFR tyrosine phosphorylation and amphiregulin expression in colonocytes. (A) Western blot analysis of phos- phorylated EGFR and EGFR in the colon. Results from 3 representative samples obtained from WT or TLR4-/- mice are shown. A total of 25 g/lane of protein was loaded per lane. The membrane was probed sequentially for phospho-EGFR and EGFR. -actin was used to show equal protein loading. (B) LPS induces the release of amphiregulin protein. SW480 cells were treated with vehicle (control) or LPS (2 g/mL) for the indicated periods. Amphiregulin concentration in supernatants was measured by enzyme-linked immunosorbent assay. Data are expressed as mean SD of relative values of expression in 3 individual experiments with triplicate samples (*P .05). (C) LPS-mediated induction of amphiregulin mRNA is TLR4-dependent. SW480 cells were transfected transiently with control siRNA or siRNA against TLR4 and then stimulated with LPS (2 g/mL) for the indicated periods of time. Untransfected control samples were not LPS treated. Levels of amphiregulin mRNA were determined by real-time PCR. The data are presented as mean SD of relative values of expression in 3 individual experiments with triplicate samples (*P .05). Inset: Western blot shows TLR4 expression with medium alone, control siRNA, and TLR4 siRNA in SW480 cells. The last bar is THP-1 cells as a positive control. (D) LPS-mediated activation of EGFR is ligand-dependent. SW480 cells were pretreated with antibodies to the ligand-binding site of EGFR, anti-amphiregulin antibody, or control immunoglobulin G for 2 hours. Subsequently, cells were stimulated with LPS (2 g/mL) for 30 minutes. Blots of whole-cell lysates (25 g/lane) were probed sequentially for phospho-EGFR and EGFR. The data are 1 representative experiment of 3 with similar results. -actin was used as an internal control for protein loading. (/) TLR4 deficiency is associated with reduced colonic mucosal production of amphiregulin. The production of amphiregulin was measured in colonic mucosa from WT (n 10) and TLR4-/- (n 8) mice. Data are expressed as mean SD (*P .05).

Article Snippet: Antibodies against hospho-EGFR (Tyr 1068, mAb) for human cells was o urchased from Cell Signaling (Beverly, MA).

Techniques: Phospho-proteomics, Expressing, Western Blot, Membrane, Control, Concentration Assay, Enzyme-linked Immunosorbent Assay, Transfection, Real-time Polymerase Chain Reaction, Positive Control, Activation Assay, Ligand Binding Assay

Journal: Gastroenterology

Article Title: Toll-like receptor-4 promotes the development of colitis-associated colorectal tumors.

doi: 10.1053/j.gastro.2007.09.008

Figure Lengend Snippet: Figure 6. Model of TLR4-mediated colon carcinogenesis. TLR4 expression is increased in chronic intestinal inflammation. TLR4 signaling in response to LPS induces Cox-2 expression and PGE2 production. PGE2 through its receptors (EP) can act in a paracrine or autocrine fashion on colonocytes to stimulate the expression and release of amphiregulin, an EGFR ligand. EGFR signaling is associated with increased proliferation of colonocytes. Likewise, TLR4 expression in tumor-associated macrophages also may respond to LPS by inducing Cox-2 and PGE2, which then may act on the epithelium to stimulate proliferation of colonocytes.

Article Snippet: Antibodies against hospho-EGFR (Tyr 1068, mAb) for human cells was o urchased from Cell Signaling (Beverly, MA).

Techniques: Expressing

Journal: Stem cells international

Article Title: Developing a Novel and Convenient Model for Investigating Sweat Gland Morphogenesis from Epidermal Stem Cells.

doi: 10.1155/2019/4254759

Figure Lengend Snippet: Figure 4: Detection of key soluble morphogens in the system of sweat gland morphogenesis and the impact of BMP receptor inhibitor and EGF receptor inhibitor on sweat gland development in vitro. BMP4 and EGF demonstrated sharped difference in the medium of the sweat gland organogenesis system. BMP receptor inhibitor could block the formation of sweat gland in this system, while EGF receptor inhibitor significantly reduced the expression of K18. (a) The variation tendency of BMP4 and EGF in the medium of system. The error bar meant the standard error of BMP4 and EGF concentrations in different systems at different time points. (b) The impact of BMP receptor inhibitor and EFG receptor inhibitor on sweat gland morphogenesis. In comparison with the control group, EGF receptor inhibitor significantly reduced the expression of K18, but glandular structure was still observed. BMP receptor inhibitor completely blocks the expression of K18, and no glandular structure was observed. All the nuclei were counterstained with DAPI (DAPI: blue; K18: red; bars = 200 μm and 50 μm; K18: cytokeratin 18; IM: light microscope; H&E: hematoxylin-eosin staining; IF: immunofluorescence staining).

Article Snippet: BMP4 and EGF concentrations in supernatant were measured using a mouse BMP4 (CSB-E04512m, Cusabio, Wuhan, Hubei, China) and EGF ELISA kit (EM014-96, ExCell Bio, Shanghai, China).

Techniques: In Vitro, Blocking Assay, Expressing, Comparison, Control, Light Microscopy, Staining

Journal: PLOS ONE

Article Title: Crystalline silica-exposed human lung epithelial cells presented enhanced anchorage-independent growth with upregulated expression of BRD4 and EZH2 in autocrine and paracrine manners

doi: 10.1371/journal.pone.0285354

Figure Lengend Snippet: (A) ELISA of EGF, TNF-α, IL-6, and TGF-β1 was performed with the culture supernatants of nonadherent NL20, BEAS-2B, and 16HBE 4–6 days after incubation with autocrine CS or BPDE CM compared with the autocrine control CM. ELISA of EGF, TNF-α, and IL-6 was also conducted with the culture supernatants 48 h after exposure to CS in adherent bronchial cell lines. Asterisks indicate a significant difference in concentration (*p < 0.05, **p < 0.01). Relative values are shown as mean ± SD from three independent experiments. (B) Anchorage-independent growth of NL20, BEAS-2B, and 16HBE cells was measured using MTT 5–6 days after the administration of recombinant human EGF, TNF-α, and IL-6. The MTT assay was performed for autocrine CS CM-incubated nonadherent NL20 and BEAS-2B cells four and five days after EGF-neutralizing antibody (NtAb) treatment, respectively, and for autocrine CS CM-incubated nonadherent 16HBE cells, five days after TNF-α NtAb treatment. The MTT assay was also performed for autocrine CS CM-incubated nonadherent bronchial cell lines five days after IL-6 NtAb administration. Asterisks indicate a significant difference in growth (*p < 0.05, **p < 0.01). Relative values are shown as mean ± SD from three independent experiments. (C) Expression of BRD4 and EZH2 in nonadherent 16HBE cells three days after the administration of recombinant human TNF-α determined using western blotting and the expression of BRD4 and EZH2 3–4 days after treatment of nonadherent NL20 and BEAS-2B cells with recombinant human EGF.

Article Snippet: Recombinant human EGF, TNF-α, TGF-β1, and IL-6 were purchased from R&D Systems (R&D Systems Inc., Minneapolis, MN, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Control, Concentration Assay, Recombinant, MTT Assay, Expressing, Western Blot

Journal: Cancer Research

Article Title: Anti–Vascular Endothelial Growth Factor Therapies as a Novel Therapeutic Approach to Treating Neurofibromatosis-Related Tumors

doi: 10.1158/0008-5472.can-09-3107

Figure Lengend Snippet: Figure 1. Characterization of a novel schwannoma line and the effect of VEGF inhibitors on these cell lines in vitro. A, wt merlin is below the detection limit both in the newly established murine schwannoma line (Nf2−/−) and in the human HEI193. Liver epithelial cells derived from the Nf2 wt mice served as a merlin-positive control. B, effect of vandetanib and bevacizumab on Nf2−/−and HEI193 signaling. Nf2-deficient or HEI193 schwannoma at density of 2.5 × 106 cells were starved overnight for 12 hours with DMEM and incubated with full medium, recombinant mouse VEGF (10 ng/mL), or human EGF (100 ng/mL) with various concentration of vandetanib or bevacizumab for 15 minutes at 37°C as indicated in the diagram. Vandetanib decreased P-EGFR, P-AKT, and P-ERK in a concentration-dependent manner, whereas bevacizumab did not affect HEI193 signaling unless at very high concentration. C, expression pattern for SEMA3, its receptors, and the VEGF receptors in murine wt Schwann cells and their Nf2−/−counterpart (top) and human Schwann cells compared with HEI193 (bottom). Loss of the Nf2 gene is concurrent with a loss of SEMA3 (b, d, f, and g in murine, all in human), NRP1, and VEGFR1 expression, whereas NRP2 and plexins retain their initial expression. VEGF receptors are not typically expressed by Schwann cells, and oncogenic transformation did not seem to change this feature in vitro. D, reintroduction of the Nf2 gene is accompanied by a reexpression of SEMA3, NRP1, and VEGFR1, supporting the hypothesis of a link between loss of merlin and a change in balance within the angiogenic pathway.

Article Snippet: The following antibodies were used: phosphorylated EGFR (P-EGFR; 1:1,000; Cell Signaling Technology), phosphorylated AKR mouse T-cell lymphoma oncogene (P-AKT; 1:1,000; Cell Signaling Technology), phosphorylated extracellular signalregulated kinase (P-ERK; 1:1,000; Cell Signaling Technology), EGFR (1:1,000; Cell Signaling Technology), AKT (1:1,000; Cell Signaling Technology), ERK1 (clone MK12, 1:1,000; BD Transduction Laboratories), and merlin (1:1,000; Santa Cruz Biotechnology).

Techniques: In Vitro, Derivative Assay, Positive Control, Incubation, Recombinant, Concentration Assay, Expressing, Transformation Assay

Journal: Lung cancer (Amsterdam, Netherlands)

Article Title: Mutant forms of EGFR promote HER2 trafficking through efficient formation of HER2-EGFR heterodimers.

doi: 10.1016/j.lungcan.2022.11.018

Figure Lengend Snippet: Fig. 1. Detection of HER2 homodimers and heterodimers with an in situ PLA in cancer cell lines. Lung or breast cancer cell lines positive for HER2 amplification (amp) and WT for EGFR (A) as well as those without HER2 amplification and either negative (B) or positive (C) for EGFR activating mutations were subjected to a PLA (green signals) for detection of HER2 homodimers or HER2-EGFR or HER2-HER3 heterodimers. The representative images were obtained by optical sectioning. Nuclei (blue signals) were stained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bars, 20 µm.

Article Snippet: The membrane was incubated overnight at 4 ◦C with primary antibodies to phosphorylated EGFR (#3777, diluted 1:1000; Cell Signaling Technology, Danvers, MA, USA), to EGFR (#4267, diluted 1:1000; Cell Signaling Technology), to HER2 (#ab134182, diluted 1:1000; Abcam, Cambridge, MA, USA), to HER3 (#sc-81455, diluted 1:200; Santa Cruz Biotechnology, Dallas, TX, USA), and to β-actin (#4970, diluted 1:1000; Cell Signaling Technology).

Techniques: In Situ, Amplification, Staining

Journal: Lung cancer (Amsterdam, Netherlands)

Article Title: Mutant forms of EGFR promote HER2 trafficking through efficient formation of HER2-EGFR heterodimers.

doi: 10.1016/j.lungcan.2022.11.018

Figure Lengend Snippet: Fig. 2. EGFR activating mutations promote HER2-EGFR heterodimerization in cancer cell lines without HER2 amplification. (A) z-Projection images were con structed by combination of optical sectioning images of multiple cells obtained by z-stacking. (B) Representative z-projection images obtained with an in situ PLA for HER2-EGFR heterodimers (green signals) in lung or breast cancer cell lines negative for HER2 amplification and either negative or positive for EGFR activating mutations. Nuclei (blue signals) were stained with DAPI. Scale bars, 20 µm. (C) Quantification of the number of HER2-EGFR heterodimers per cell from images as in (B). Data are means + SEM (left, n = 50 cells) or means ± SD (right, n = 6 cell lines). *p < 0.05 (unpaired Student’s t test). (D) Immunoblot analysis of HER2 and EGFR in lung and breast cancer cell lines. β-actin was examined as a loading control. (E) Relative MFI ratios for expression of HER2 and EGFR at the cell surface determined by flow cytometric analysis. Data are means + SD from three independent experiments. (F) Representative z-projection images obtained with an in situ PLA performed as in (B) for HER2-EGFR heterodimers in MCF7 cells infected with retroviruses encoding WT or mutant (del19 or L858R) forms of EGFR. (G) Number of HER2-EGFR heterodimers per cell for cells as in (F). Data are means + SEM (n = 50 cells). ****p < 0.0001 (one-way ANOVA). All data are representative of or from three independent experiments.

Article Snippet: The membrane was incubated overnight at 4 ◦C with primary antibodies to phosphorylated EGFR (#3777, diluted 1:1000; Cell Signaling Technology, Danvers, MA, USA), to EGFR (#4267, diluted 1:1000; Cell Signaling Technology), to HER2 (#ab134182, diluted 1:1000; Abcam, Cambridge, MA, USA), to HER3 (#sc-81455, diluted 1:200; Santa Cruz Biotechnology, Dallas, TX, USA), and to β-actin (#4970, diluted 1:1000; Cell Signaling Technology).

Techniques: Amplification, In Situ, Staining, Western Blot, Control, Expressing, Infection, Mutagenesis

Journal: Lung cancer (Amsterdam, Netherlands)

Article Title: Mutant forms of EGFR promote HER2 trafficking through efficient formation of HER2-EGFR heterodimers.

doi: 10.1016/j.lungcan.2022.11.018

Figure Lengend Snippet: Fig. 3. HER2 is rapidly internalized in cancer cell lines negative for HER2 amplification. HER2 amplification–negative lung cancer cell lines (A), HER2 amplifi cation–positive cell lines (B), and MCF7 cells infected with retroviruses encoding WT or mutant (del19 or L858R) forms of EGFR (C) were exposed to Tz-555 at 4 ◦C for 30 min, washed, and incubated at 37 ◦C beginning at time 0 min for live-cell imaging of Tz-555–labeled HER2 (yellow). Data are representative of two inde pendent experiments. Scale bars, 10 μm.

Article Snippet: The membrane was incubated overnight at 4 ◦C with primary antibodies to phosphorylated EGFR (#3777, diluted 1:1000; Cell Signaling Technology, Danvers, MA, USA), to EGFR (#4267, diluted 1:1000; Cell Signaling Technology), to HER2 (#ab134182, diluted 1:1000; Abcam, Cambridge, MA, USA), to HER3 (#sc-81455, diluted 1:200; Santa Cruz Biotechnology, Dallas, TX, USA), and to β-actin (#4970, diluted 1:1000; Cell Signaling Technology).

Techniques: Amplification, Infection, Mutagenesis, Incubation, Live Cell Imaging, Labeling

Journal: Lung cancer (Amsterdam, Netherlands)

Article Title: Mutant forms of EGFR promote HER2 trafficking through efficient formation of HER2-EGFR heterodimers.

doi: 10.1016/j.lungcan.2022.11.018

Figure Lengend Snippet: Fig. 4. HER2 is internalized together with EGFR in HER2 amplification–negative cancer cell lines. A549 and PC-9 cells were exposed to Tz-555 and Cmab-647 at 4 ◦C for 30 min, washed, and incubated at 37 ◦C beginning at time 0 min for live-cell imaging of Tz-555–labeled HER2 (yellow) and Cmab-647–labeled EGFR (purple). Colocalization of Tz-555 and Cmab-647 signals is indicated in white in the merged images, with the boxed regions of the upper merged panels being shown at higher magnification in the lower panels. Data are representative of two independent experiments. Scale bars, 10 μm.

Article Snippet: The membrane was incubated overnight at 4 ◦C with primary antibodies to phosphorylated EGFR (#3777, diluted 1:1000; Cell Signaling Technology, Danvers, MA, USA), to EGFR (#4267, diluted 1:1000; Cell Signaling Technology), to HER2 (#ab134182, diluted 1:1000; Abcam, Cambridge, MA, USA), to HER3 (#sc-81455, diluted 1:200; Santa Cruz Biotechnology, Dallas, TX, USA), and to β-actin (#4970, diluted 1:1000; Cell Signaling Technology).

Techniques: Amplification, Incubation, Live Cell Imaging, Labeling

Journal: Lung cancer (Amsterdam, Netherlands)

Article Title: Mutant forms of EGFR promote HER2 trafficking through efficient formation of HER2-EGFR heterodimers.

doi: 10.1016/j.lungcan.2022.11.018

Figure Lengend Snippet: Fig. 6. EGFR activating mutations increase cell sensitivity to T-DM1. (A) Relation between the rela tive MFI ratio for HER2 at the cell surface and the median inhibitory concentration (IC50) value for cytotoxicity of T-DM1 for cancer cell lines classified according to HER2 amplification and EGFR activating mutation status. Data for MCF7 cells are not shown because the IC50 value was > 500 µg/ml. Data are means of triplicates from one experiment and are representative of three independent experiments. (B) Percentage cell viability for MCF7 cells stably expressing WT or mutant (del19 or L858R) forms of EGFR and exposed to T-DM1 (100 µg/ml) for 72 h. Data are means + SEM (n = 3 triplicates from one experiment, with the data being representative of three independent experiments). **p < 0.01, ***p < 0.001 (one-way ANOVA).

Article Snippet: The membrane was incubated overnight at 4 ◦C with primary antibodies to phosphorylated EGFR (#3777, diluted 1:1000; Cell Signaling Technology, Danvers, MA, USA), to EGFR (#4267, diluted 1:1000; Cell Signaling Technology), to HER2 (#ab134182, diluted 1:1000; Abcam, Cambridge, MA, USA), to HER3 (#sc-81455, diluted 1:200; Santa Cruz Biotechnology, Dallas, TX, USA), and to β-actin (#4970, diluted 1:1000; Cell Signaling Technology).

Techniques: Concentration Assay, Amplification, Mutagenesis, Stable Transfection, Expressing

Journal: Lung cancer (Amsterdam, Netherlands)

Article Title: Mutant forms of EGFR promote HER2 trafficking through efficient formation of HER2-EGFR heterodimers.

doi: 10.1016/j.lungcan.2022.11.018

Figure Lengend Snippet: Fig. 5. Internalized HER2 is efficiently transferred to lysosomes in EGFR mutation–positive cancer cells. (A) A549 and PC-9 cells were exposed to chloroquine, labeled with Tz-555 for 30 min at 4 ◦C, and then incubated for 2 h at 37 ◦C in the presence of chloroquine, after which they were stained with antibodies to LAMP1 and both Tz-555 (yellow) and LAMP1 (purple) signals were detected by fluorescence microscopy. Colocalization of Tz-555 and LAMP1 signals is indicated in white in the merged panels. Dashed lines indicate cell boundaries. Scale bars, 10 µm. (B) Quantification of Tz-555 colocalization with LAMP1 in cells as in (A). (C) MCF7 cells stably expressing WT or mutant (del19 or L858R) forms of EGFR were analyzed as in (A). Scale bars, 10 µm. (D) Quantification of Tz-555 colocalization with LAMP1 in cells as in (C). The representative images in (A) and (C) were obtained by optical sectioning. The quantitative data (n = 9 fields) in (B) and (D) are presented as box plots, with the boxes representing the median and 25th and 75th percentiles and the whiskers extending to maximum and minimum values. *p < 0.05, ***p < 0.001 (Mann-Whitney test); ****p < 0.0001 (unpaired Student’s t test). All data are representative of three independent experiments.

Article Snippet: The membrane was incubated overnight at 4 ◦C with primary antibodies to phosphorylated EGFR (#3777, diluted 1:1000; Cell Signaling Technology, Danvers, MA, USA), to EGFR (#4267, diluted 1:1000; Cell Signaling Technology), to HER2 (#ab134182, diluted 1:1000; Abcam, Cambridge, MA, USA), to HER3 (#sc-81455, diluted 1:200; Santa Cruz Biotechnology, Dallas, TX, USA), and to β-actin (#4970, diluted 1:1000; Cell Signaling Technology).

Techniques: Mutagenesis, Labeling, Incubation, Staining, Fluorescence, Microscopy, Stable Transfection, Expressing, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: Aloysia Citrodora Essential Oil Inhibits Melanoma Cell Growth and Migration by Targeting HB-EGF-EGFR Signaling

doi: 10.3390/ijms22158151

Figure Lengend Snippet: ALOC-EO targets EGFR and ERK1/2. ( A ) Immunoblot for phosphorylated murine EGFR in B16F10 cells treated with either DMSO or ALOC-EO after 12 h. Right panel: The histogram represents the signal intensity of the indicated protein bands in arbitrary units after normalization with the signal intensity of β-actin internal control for each sample. Values are means and SEM of two independent experiments. ( B ) Representative immunoblot for phosphorylated murine EGFR and loading control β-actin in tumor cell lysates from control and ALOC-EO-treated mice. Right panel: The histogram represents the signal intensity of the indicated protein bands in arbitrary units after normalization with the signal intensity of β-actin internal control for each sample. Values are means and SEM of two independent experiments. ( C ) EGFR overexpression (EGFR OE) was confirmed in B16F10 cells and mock controls by qPCR, and the fold change in EGFR expression compared to expression of β-actin is given (n = 3). ( D ) EGFR phosphorylation was performed in cell lysates of EGFR OE and mock cells by Western blotting. Right panel: The histogram represents the signal intensity of the indicated protein bands in arbitrary units after normalization with the signal intensity of β-actin internal control for each sample. Values are means and SEM of two independent experiments. ( E ) EGFR OE and mock cells were treated with/without ALOC-EO. Cells were counted after 24 h in culture (n = 6/group). ( F ) B16F10 cells were treated with/without rec. HB-EGF in the presence or absence of ALOC-OC. Cells were counted 24 h later (n = 6/group). ( G ) Murine MMP7 , MMP9 , ADAM9 , and HB-EGF expression in EGFR OE and mock B16F10 cells treated with or without HB-EGF in the presence or absence of ALOC-EO by qPCR. Transcript levels were normalized to β-actin. +/− SEM. * p < 0.05; ** p < 0.01 (Student’s t -test).

Article Snippet: Cell lysates (2–50 μg proteins) were applied on 12% acrylamide gel, transferred to PVDF membrane (Millipore, Immobilon), and then probed with one of the following primary antibodies (all mouse IgG, 1 μg/mL) overnight at 4 °C: C-terminal cytoplasmic domain of HB-EGF of mouse origin (Santa Cruz Biotech, Santa Cruz, CA, USA, sc-1414), MMP7 (Santa Cruz Biotech, Santa Cruz, CA, USA, sc-515702), β-actin (Cell Signaling, #4967), total p44/42 MAPK (Erk1/2)(Cell Signaling, #9102), p-EGFR (Santa Cruz Biotech, sc-57545), MMP9 (Chemicom, AB19047)), ERK1/2 (Cell signaling, 4370S), BAX2 (Cell signaling, Danvers, MA, USA, #2772S).

Techniques: Western Blot, Control, Over Expression, Expressing, Phospho-proteomics

Journal: International Journal of Molecular Sciences

Article Title: Aloysia Citrodora Essential Oil Inhibits Melanoma Cell Growth and Migration by Targeting HB-EGF-EGFR Signaling

doi: 10.3390/ijms22158151

Figure Lengend Snippet: Chemotherapy-induced HB-EGF-EGFR upregulation is blocked by ALOC-EO and improves chemo-sensitivity. ( A ) Fold change expression of murine HB-EGF and ( B ) EGFR in B16F10 cells treated with indicated concentrations of the antitumor drugs bortezomib or doxorubicin, as a carrier by qPCR (A; n = 3/group). ( C – E ) HB-EGF expression was determined after ALOC-EO treatment for 24 h in B16F10 cells and tumor tissues retrieved from tumor-bearing mice treated with or without ALOC-EO at day 12 by qPCR (C; n = 3/group) by Western blotting analysis ( D , E ). Right panel: The histograms represent the signal intensity of the indicated protein bands in arbitrary units after normalization with the signal intensity of β-actin internal control for each sample. Values are means and SEM of two independent experiments. ( F – H ) B16F10 cells were treated with BTZ ( G ) or doxorubicin ( F , H ) at indicated concentration in the presence or absence of 100 µg/mL ALOC-EO. ( F ) Fold expression of MMP7/9 , and ADAM9 in cells treated with indicated drug combinations as determined by qPCR. ( G , H ) Viable cells were counted after 24 h, using the trypan blue exclusion assay (n = 6). The results of three independent experiments performed in triplicates are expressed as fold change according to 2-ΔΔCT method using β-actin as calibrator. Transcript levels were normalized to β-actin (n = 3/group). ( I ) Proposed mode of action for ALOC-EC in melanoma: ALOC-EO can block EGFR and ERK1/2 phosphorylation, leading to the reduced expression of MMP7/9 and ADAM9. Not shown in this study is that these proteases are known to enhance the shedding of the EGFR ligand HB-EGF. Abbreviations: ALOC-EO, Aloysia citrodora essential oil; EGFR, epidermal growth factor receptor; ERK1/2, extracellular signal-regulated kinase; MMP7, matrix metalloproteinase-7, matrix metalloproteinase9 (MMP9), a disintegrin and metalloproteinase domain-containing protein 9 (ADAM9). * p < 0.05; ** p < 0.01.

Article Snippet: Cell lysates (2–50 μg proteins) were applied on 12% acrylamide gel, transferred to PVDF membrane (Millipore, Immobilon), and then probed with one of the following primary antibodies (all mouse IgG, 1 μg/mL) overnight at 4 °C: C-terminal cytoplasmic domain of HB-EGF of mouse origin (Santa Cruz Biotech, Santa Cruz, CA, USA, sc-1414), MMP7 (Santa Cruz Biotech, Santa Cruz, CA, USA, sc-515702), β-actin (Cell Signaling, #4967), total p44/42 MAPK (Erk1/2)(Cell Signaling, #9102), p-EGFR (Santa Cruz Biotech, sc-57545), MMP9 (Chemicom, AB19047)), ERK1/2 (Cell signaling, 4370S), BAX2 (Cell signaling, Danvers, MA, USA, #2772S).

Techniques: Expressing, Western Blot, Control, Concentration Assay, Trypan Blue Exclusion Assay, Blocking Assay, Phospho-proteomics

Journal: ACS chemical biology

Article Title: High Throughput Screens to Identify Autophagy Inducers that Function by Disrupting Beclin 1/Bcl-2 Binding

doi: 10.1021/acschembio.8b00421

Figure Lengend Snippet: Effects of Beclin 1/Bcl-2 binding disruptors on autophagy and cell death. (A-B) Representative images (A) or quantitation (B) of GFP-LC3-positive puncta in HeLa/GFP-LC3 cells treated with DMSO or indicated compound. 20 μM compound +/− 100 nM Baf A1, 24 h. Bars represent mean ± s.e.m. of triplicate samples (>100 cells analyzed per sample). Similar results were observed in three independent experiments. ***p<0.001; Mann–Whitney U test. Scale bars, 1 μm. (C) Western blot analysis of LC3 in HeLa cells treated with indicated concentration of indicated compound for 24 h in the presence or absence of 100 nM Baf A1. (D) Effects of indicated compounds at indicated concentrations on HeLa cell apoptosis as assessed by western blot detection of cleaved PARP.

Article Snippet: The following antibodies were used for western blot analyses: goat polyclonal anti-luciferase (Promega G7451, 1:1000 dilution), rabbit anti-Beclin 1 (Santa Cruz sc-11427, 1:500 dilution), HRP-conjugated anti-Myc (Santa Cruz SC-40 HRP, 1:100 dilution), rabbit anti-Bax (Cell Signaling Technology #5023, 1:500 dilution), rabbit anti-LC3 (Novus NB100–2220, 1:2000 dilution), mouse anti-p62 (Abnova #H00008878-M01, 1:20000 dilution), rabbit anti-Bim (Cell Signaling Technology #2933, 1:1000 dilution), rabbit anti-cleaved PARP (Cell Signaling Technology #9451, 1:1000 dilution) and HRP-conjugated anti-actin (Santa Cruz sc-47778-HRP, 1:5000 dilution).

Techniques: Binding Assay, Quantitation Assay, MANN-WHITNEY, Western Blot, Concentration Assay

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Biological and clinical impact of membrane EGFR expression in a subgroup of OC patients from the phase IV ovarian cancer MITO-16A/MANGO-OV2A trial.

doi: 10.1186/s13046-023-02651-y

Figure Lengend Snippet: Fig. 2 Kaplan-Meyer analysis evaluating the progression free survival (PFS) and the overall survival (OS) of patients from the MITO16a-MaNGO-OV2i stratified according to EGFR membrane expression by immunohistochemical staining

Article Snippet: The primary antibodies used were: mouse anti-phosphorylated EGFR (Tyrosine 1068) (1H12, Cell Signaling.

Techniques: Membrane, Expressing, Immunohistochemical staining, Staining

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Biological and clinical impact of membrane EGFR expression in a subgroup of OC patients from the phase IV ovarian cancer MITO-16A/MANGO-OV2A trial.

doi: 10.1186/s13046-023-02651-y

Figure Lengend Snippet: Fig. 1 Representative images of OC sections stained with the anti-EGFR antibody. C, cytoplasmic staining; NEG, no staining. Percentage of the clear cell membrane staining is reported below pictures. A Representative images of an entire section within the TMA is reported. Black empty box, area shown in panel B. Bar, 200 μm. B Higher magnification of each section in panel A

Article Snippet: The primary antibodies used were: mouse anti-phosphorylated EGFR (Tyrosine 1068) (1H12, Cell Signaling.

Techniques: Staining, Membrane

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Biological and clinical impact of membrane EGFR expression in a subgroup of OC patients from the phase IV ovarian cancer MITO-16A/MANGO-OV2A trial.

doi: 10.1186/s13046-023-02651-y

Figure Lengend Snippet: Fig. 3 A Graphical representation of the mean GSVA scores for the EGFR-related gene sets (see Table 3 for GSVA scores and GSEA). The red line highlights the 0 score. Different dots’ colors represent different gene sets, as reported. For the corresponding GSEA nomenclature refers to Table 4. B Graphical representation of the overlap among the gene sets significantly enriched in MM staining subgroup defined in the MITO16a-MaNGO-OV2 trial. The three gene sets (a), (d) and (e) were selected for further analysis since they include all the EGFR-related genes. Each black line represents a gene; for each gene set the number of genes is reported at the bottom. The names of the gene sets are reported below the scheme

Article Snippet: The primary antibodies used were: mouse anti-phosphorylated EGFR (Tyrosine 1068) (1H12, Cell Signaling.

Techniques: Staining

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Biological and clinical impact of membrane EGFR expression in a subgroup of OC patients from the phase IV ovarian cancer MITO-16A/MANGO-OV2A trial.

doi: 10.1186/s13046-023-02651-y

Figure Lengend Snippet: Fig. 4 Graphical representation of the genes/functions predicted by IPA performed using the differential gene expression derived by comparing MM vs M and MO groups of patients. IPA was run using the log fold changes of the genes up- or down-modulated in MM vs the other two subgroups and included in the EGFR-related gene sets reported in Table 4. See Methods for details of the analysis. The name of each gene set is reported above each scheme. Panel in the right side, prediction legends

Article Snippet: The primary antibodies used were: mouse anti-phosphorylated EGFR (Tyrosine 1068) (1H12, Cell Signaling.

Techniques: Gene Expression, Derivative Assay

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Biological and clinical impact of membrane EGFR expression in a subgroup of OC patients from the phase IV ovarian cancer MITO-16A/MANGO-OV2A trial.

doi: 10.1186/s13046-023-02651-y

Figure Lengend Snippet: Fig. 6 A Western blotting for EGFR and AXL expressions in lysates from OC cell lines representative of HGSOCs or non-HGSOCs cell lines [34]. SKOV3 and OVCAR5 cells co-expressed both EGFR and AXL. B Confocal immunofluorescence showing EGFR and Axl expressions; only SKOV3 showed both EGFR (green) and AXL (red) expressions on the cell membrane with several regions of co-localization (white box indicates one of those regions). In OVCAR5 cells only EGFR is clearly expressed on the membrane. SKOV3 was chosen for further analysis. Upper, merge staining; lower. single staining, Nuclei were stained with DAPI. C upper. Western blotting of lysated from SKOV3 cells stimulated with GAS6 or EGF alone or in combination; lower, densitometric analysis for phoshorylated (P-EGFR and P–AXL) or total RTKs (EGFR and AXL). As expected, the total amount of EGFR decreased upon ligand stimulation [35]. No changes in the amount of AXL were detected upon GAS6 stimulation. D Erlotinib susceptibility of AXL silenced SKOV3 cells. Upper, western blotting showing the amount of silenced AXL in SKOV3 lysates upon transfection with two different siRNAs (#1 and #2). Lower, viability of SKOV3 cells treated with control siRNA (siCO) or with specific Axl siRNAs (siAxl#1 and #2) and then treated with erlotinib at different concentration. Statistical evaluation by ANOVA, p ≤ 0.001. Refer to Methods section for detailed procedure. The table below reports the IC50 values of siRNA transfected cells. The experiments were performed at least three times

Article Snippet: The primary antibodies used were: mouse anti-phosphorylated EGFR (Tyrosine 1068) (1H12, Cell Signaling.

Techniques: Western Blot, Immunofluorescence, Membrane, Staining, Transfection, Control, Concentration Assay