Journal: PLoS ONE

Article Title: A new sensitive and fast assay for the detection of EGFR mutations in liquid biopsies

doi: 10.1371/journal.pone.0253687

Figure Lengend Snippet: Median age was 65.5 years for cohort I and 69.5 years for cohort II at the time of routine ctDNA EGFR analyses.

Article Snippet: In our setup, the SensiScreen ® EGFR Liquid assay platform (PentaBase) outperformed the Therascreen ® platform (Qiagen) and showed 100% agreement at the sequence level to the IOT ® Oncomine cell-free nucleic acids assay (Thermo Fisher Scientific).

Techniques:

Journal: PLoS ONE

Article Title: A new sensitive and fast assay for the detection of EGFR mutations in liquid biopsies

doi: 10.1371/journal.pone.0253687

Figure Lengend Snippet: PCR curves obtained when challenging assays with cell lines or plasmids containing the indicated EGFR mutations. Plasmids were diluted 1:1 in WT gDNA to ensure a total template amount of 1600 copies. Reference assay PCR curves are represented by heavy coloured lines and mutation specific assay PCR curves by light coloured lines as indicated in figure legends. Abbreviations: Ct, Cycle threshold; EGFR, Epidermal growth factor receptor.

Article Snippet: In our setup, the SensiScreen ® EGFR Liquid assay platform (PentaBase) outperformed the Therascreen ® platform (Qiagen) and showed 100% agreement at the sequence level to the IOT ® Oncomine cell-free nucleic acids assay (Thermo Fisher Scientific).

Techniques: Mutagenesis

Journal: PLoS ONE

Article Title: A new sensitive and fast assay for the detection of EGFR mutations in liquid biopsies

doi: 10.1371/journal.pone.0253687

Figure Lengend Snippet: A. Histograms showing the frequency and distribution of mutant copies detected in 102 replicates for assays. B. Boxplot showing the collectively recovered number of copies by the three assays and thereby the overall detected copy number by assays. Abbreviations: EGFR, Epidermal growth factor receptor.

Article Snippet: In our setup, the SensiScreen ® EGFR Liquid assay platform (PentaBase) outperformed the Therascreen ® platform (Qiagen) and showed 100% agreement at the sequence level to the IOT ® Oncomine cell-free nucleic acids assay (Thermo Fisher Scientific).

Techniques: Mutagenesis

Journal: PLoS ONE

Article Title: A new sensitive and fast assay for the detection of EGFR mutations in liquid biopsies

doi: 10.1371/journal.pone.0253687

Figure Lengend Snippet: Repeatability and accuracy evaluation of SensiScreen ® EGFR Liquid assays: Number of copies mutated for Exon 19 deletion, T790M or L858R.

Article Snippet: In our setup, the SensiScreen ® EGFR Liquid assay platform (PentaBase) outperformed the Therascreen ® platform (Qiagen) and showed 100% agreement at the sequence level to the IOT ® Oncomine cell-free nucleic acids assay (Thermo Fisher Scientific).

Techniques:

Journal: PLoS ONE

Article Title: A new sensitive and fast assay for the detection of EGFR mutations in liquid biopsies

doi: 10.1371/journal.pone.0253687

Figure Lengend Snippet: Graphs describing the LoB evaluation based on the characterization of repeated testing of cfDNA pooled from 58 healthy donors A. Curves representing the T790M LoB results. B. Curves representing the L858R LoB results. C. Curves representing the EGFR exon 19 deletion LoB results.

Article Snippet: In our setup, the SensiScreen ® EGFR Liquid assay platform (PentaBase) outperformed the Therascreen ® platform (Qiagen) and showed 100% agreement at the sequence level to the IOT ® Oncomine cell-free nucleic acids assay (Thermo Fisher Scientific).

Techniques:

Journal: PLoS ONE

Article Title: A new sensitive and fast assay for the detection of EGFR mutations in liquid biopsies

doi: 10.1371/journal.pone.0253687

Figure Lengend Snippet: Overview of the EGFR phenotypes detected by the Therascreen ® (QIAGEN) versus SensiScreen ® EGFR Liquid assay (Pentabase) and IOT ® Oncomine cell-free nucleic acids assay (Thermo Fisher Scientific) using cohort I and agreement or disagreement between platforms (N = 34).

Article Snippet: In our setup, the SensiScreen ® EGFR Liquid assay platform (PentaBase) outperformed the Therascreen ® platform (Qiagen) and showed 100% agreement at the sequence level to the IOT ® Oncomine cell-free nucleic acids assay (Thermo Fisher Scientific).

Techniques:

Journal: PLoS ONE

Article Title: A new sensitive and fast assay for the detection of EGFR mutations in liquid biopsies

doi: 10.1371/journal.pone.0253687

Figure Lengend Snippet: Overview of the EGFR phenotypes detected by the SensiScreen ® EGFR Liquid assay (PentaBase ApS) versus the ctEGFR Mutation Detection Kit (EntroGen) using cohort II and agreement or disagreement between platforms (N = 34).

Article Snippet: In our setup, the SensiScreen ® EGFR Liquid assay platform (PentaBase) outperformed the Therascreen ® platform (Qiagen) and showed 100% agreement at the sequence level to the IOT ® Oncomine cell-free nucleic acids assay (Thermo Fisher Scientific).

Techniques: Mutagenesis

Journal: Communications Biology

Article Title: DNAJC5 facilitates the proliferation and migration of lung adenocarcinoma cells by augmenting EGFR trafficking

doi: 10.1038/s42003-025-08191-9

Figure Lengend Snippet: A Silver staining was conducted to exhibit DNAJC5 interacted proteins for Mass spectrum analysis. B The results of the LC-MS/MS analysis for the identification of potential specific DNAJC5-binding proteins were presented in the Venn diagram. C The list of the potential unique proteins with high scores that interacted with DNAJC5 via LC-MS/MS analysis. D , E The indicated plasmids expressed exogenous EGFR and DNAJC5 were co-transfected into HEK293T cells, and cell lysates were immunoprecipitated and subjected to western blot. F , G Lysates from A549 cells and PC9 cells were pulled down with anti-DNAJC5 followed by western blot. H The domains of EGFR responsible for the DNAJC5 interaction with EGFR were mapped. Schematic illustration of the domains of EGFR (up). Lysates from HEK293T cells co-transfected with the indicated plasmids were incubated with an anti-Flag antibody. The immunoprecipitate (IP) and lysates were probed with the indicated antibodies (down). I The domains of DNAJC5 binding EGFR were mapped. Schematic illustration of the domains of DNAJC5 (up). Lysates from HEK293T cells co-transfected with the indicated plasmids were immunoprecipitated and subjected to western blot (down). J The indicated plasmids expressed exogenous EGFR mutants and DNAJC5 were co-transfected into HEK293T cells, and cell lysates were immunoprecipitated and subjected to western blot.

Article Snippet: The primary antibodies were used as bellow: EGFR (CST, 4267), p-EGFR (CST, #3777), AKT (CST, #4691S), ERK (CST, #4695), p-AKT (CST, #4060S), p-ERK (CST, #4695), GFP (Sigma, G1544), DNAJC5 (Millipore, AB1576), Flag (Sigma, F3165), GAPDH (Millipore, MAB374), HA (CST, 3724S).

Techniques: Silver Staining, Liquid Chromatography with Mass Spectroscopy, Binding Assay, Transfection, Immunoprecipitation, Western Blot, Incubation

Journal: Communications Biology

Article Title: DNAJC5 facilitates the proliferation and migration of lung adenocarcinoma cells by augmenting EGFR trafficking

doi: 10.1038/s42003-025-08191-9

Figure Lengend Snippet: A A549 cells overexpressing DNAJC5 and control cells were subjected to overnight serum starvation and then treated with varying concentrations of EGF for 15 minutes. The protein levels of EGFR and phosphorylated EGFR (Tyr1068) were analyzed using western blotting. B A549 cells overexpressing DNAJC5 and control cells were subjected to overnight serum starvation followed by treatment with EGF (10 ng/ml) for different durations. C H1299 cells with knockdown of DNAJC5 expression and control cells were subjected to overnight serum starvation, followed by treatment with EGF (10 ng/ml) for 15 minutes. The protein levels of EGFR and phosphorylated EGFR (Tyr1068) were analyzed using western blotting. D H1299 cells with knockdown of DNAJC5 expression and control cells were subjected to overnight serum starvation, followed by treatment with EGF (10 ng/ml) for different durations. E , F DNAJC5 strengthens AKT and ERK signaling pathways. Cells overexpressing DNAJC5 ( E ), or those in which DNAJC5 was interfered with ( F ), underwent overnight serum starvation before being treated with EGF (10 ng/ml) for different durations. The protein levels of AKT, phosphorylated AKT, ERK, and phosphorylated ERK were analyzed using western blotting.

Article Snippet: The primary antibodies were used as bellow: EGFR (CST, 4267), p-EGFR (CST, #3777), AKT (CST, #4691S), ERK (CST, #4695), p-AKT (CST, #4060S), p-ERK (CST, #4695), GFP (Sigma, G1544), DNAJC5 (Millipore, AB1576), Flag (Sigma, F3165), GAPDH (Millipore, MAB374), HA (CST, 3724S).

Techniques: Control, Western Blot, Knockdown, Expressing, Protein-Protein interactions

Journal: Communications Biology

Article Title: DNAJC5 facilitates the proliferation and migration of lung adenocarcinoma cells by augmenting EGFR trafficking

doi: 10.1038/s42003-025-08191-9

Figure Lengend Snippet: A Immunofluorescence analysis was performed on serum-starved overnight A549 cells overexpressing DNAJC5 or control A549 cells, with or without treatment with EGF (10 ng/ml) for 15 minutes. Co-localization of EGFR and EEA1 was examined. B The degree of colocalization between EGFR and EEA1 was quantified using Pearson’s coefficient. C Immunofluorescence analysis was conducted on serum-starved overnight H1299 cells with knockdown of DNAJC5 or control cells, with or without treatment with EGF (10 ng/ml) for 15 minutes. Co-localization of EGFR and EEA1 was examined. D The degree of colocalization between EGFR and EEA1 was quantified using Pearson’s coefficient. E , F Overexpression of DNAJC5 promoted the endocytosis of EGFR, while knockdown of DNAJC5 suppressed it. To assess internalized EGFR levels, Sulfo-NHS-SS-biotin labeling was performed on ice followed by stimulation with EGF at 37°C for various durations. Cell lysates were immunoprecipitated and subjected to western blotting. Image J software was used to analyze the gray value corresponding to internalized EGFR. G Overexpression of DNAJC5 facilitated the recycling process of EGFR. In A549 cells, Sulfo-NHS-SS-biotin labeling on ice followed by stimulation with EGF at 37 °C for 15 minutes allowed surface biotin cleavage by MesNa. Subsequent incubation with EGF at 37 °C for different time periods induced the recycling process in which internalized EGFR returned to the cell surface. Cell lysates were immunoprecipitated and subjected to western blotting. Image J software was used to analyze the gray value corresponding to internalized EGFR.

Article Snippet: The primary antibodies were used as bellow: EGFR (CST, 4267), p-EGFR (CST, #3777), AKT (CST, #4691S), ERK (CST, #4695), p-AKT (CST, #4060S), p-ERK (CST, #4695), GFP (Sigma, G1544), DNAJC5 (Millipore, AB1576), Flag (Sigma, F3165), GAPDH (Millipore, MAB374), HA (CST, 3724S).

Techniques: Immunofluorescence, Control, Knockdown, Over Expression, Labeling, Immunoprecipitation, Western Blot, Software, Incubation

Journal: Communications Biology

Article Title: DNAJC5 facilitates the proliferation and migration of lung adenocarcinoma cells by augmenting EGFR trafficking

doi: 10.1038/s42003-025-08191-9

Figure Lengend Snippet: A – D DNAJC5 interacts with various components of the AP2 complex. Plasmids expressing exogenous AP2M1 ( A ), AP2S1 ( B ), or AP2A1 ( C , D ) were co-transfected with plasmids expressing DNAJC5 into HEK293T cells. The cell lysates were then immunoprecipitated and subjected to western blot analysis. E DNAJC5 strengthens the binding of EGFR to AP2A1. Lysates from cells co-transfected with indicated plasmids were immunoprecipitated and analyzed by western blotting. F – K Knockdown DNAJC5 attenuated the ability of DNAJC5 promoting LUAD cell proliferation and migration. F AP2A1 was interfered in DNAJC5-overexpressed A549 cells and protein levels was detected by western blot. G CCK8 cell viability assays are presented as indicated. H , I Colony formation assays are shown as indicated and the results were quantified. J , K Cell migration rates were detected by Trans-well assays, and the results were quantified.

Article Snippet: The primary antibodies were used as bellow: EGFR (CST, 4267), p-EGFR (CST, #3777), AKT (CST, #4691S), ERK (CST, #4695), p-AKT (CST, #4060S), p-ERK (CST, #4695), GFP (Sigma, G1544), DNAJC5 (Millipore, AB1576), Flag (Sigma, F3165), GAPDH (Millipore, MAB374), HA (CST, 3724S).

Techniques: Expressing, Transfection, Immunoprecipitation, Western Blot, Binding Assay, Knockdown, Migration

Journal: Cancer Research

Article Title: Insulin-like Growth Factor-I Receptor/Human Epidermal Growth Factor Receptor 2 Heterodimerization Contributes to Trastuzumab Resistance of Breast Cancer Cells

doi: 10.1158/0008-5472.can-04-3841

Figure Lengend Snippet: Figure 1. Heterodimerization between IGF-IR and HER-2 occurs uniquely in trastuzumab-resistant cells. A, relative levels of total IGF-IR were assessed in parental cells and resistant pools by immunoblotting. B, IGF-IR was immunoprecipitated (200 Ag) from whole-cell lysates of parental and resistant cells and immunoblotted for phosphotyrosine (monoclonal PT66). A 90-kDa band corresponding to phospho-IGF-IR (p-IGF-IR) was detected in all immunoprecipitates. A 185-kDa phosphoprotein (asterisk) was also observed and was found exclusively in trastuzumab-resistant cells (pool 1 and pool 2). Immunoblotting for IGF-IR confirmed equal levels of IGF-IR in all immunoprecipitates. C, the 185-kDa band found exclusively in the resistant cells was manually cut from a silver-stained gel. After trypsin digestion, peptides were extracted and analyzed by LC-MS/MS. Tryptic peptide coverage map; boldfaced sequences, identified by MS and matched HER-2 (gi|4758298|). D, to confirm the HER-2 identity, IGF-IR was immunoprecipitated from whole-cell lysates and immunoblotted for HER-2. Conversely, immunoprecipitation of HER-2 with immunoblotting for IGF-IR reveals the same exclusivity of the IGF-IR/HER-2 interaction in resistant cells. E, to determine the specificity of IGF-IR interaction with HER-2 in resistant cells, IGF-IR was immunoprecipitated from parental and resistant cells and immunoblotted for EGFR (a related family member of HER-2), integrin h3 (a nontyrosine kinase receptor membrane protein), or PDGFR (a non-EGFR tyrosine kinase receptor). Interaction between IGF-IR and EGFR was not observed in any of the immunoprecipitates. (Faint bands observed in resistant cell immunoprecipitates likely represent cross-reactivity of the EGFR antibody with HER-2.) As a control for EGFR immunoblotting, HER-2 was immunoprecipitated and immunoblotted for EGFR. As expected, EGFR was detected in the HER-2 immunoprecipitates of all lysates. IGF-IR interacted with integrin h3 in all cells but not any more so in resistant cells versus parental cells. PDGFR was not detected in any of the IGF-IR immunoprecipitates. (The control for PDGFR was 3T3 cell lysate.) F, whole-cell lysates from parental and resistant cells were immunoblotted for total phosphotyrosine (monoclonal PT66), HER-2 phosphorylated on tyrosine (Y) 1248, and total HER-2, all of which were expressed at similar levels in parental and resistant cells.

Article Snippet: Total protein extracts (50 Ag) were immunoblotted using the following antibodies at the indicated dilutions: polyclonal antibodies for EGF receptor (EGFR), integrin h3, and platelet-derived growth factor receptor (PDGFR) at 1:1000 (Santa Cruz Biotechnology, Santa Cruz, CA); polyclonal p-Tyr1162/ 1163 IR/IGF-IR at 1:500 (BioSource); HER-2 monoclonal Ab-3 at 1:1,000 (Oncogene Research Products); p-Ser473-Akt (monoclonal 587F11) and polyclonal antibodies against IGF-IR, insulin receptor substrate 1 (IRS-1), Akt, p-Thr202/Tyr204 p42/p44 mitogen-activated protein kinase (MAPK), and total p42/p44 MAPK at 1:1,000 (Cell Signaling); polyclonal against p-Ser789-IRS-1 and polyclonal against p-Tyr1248-HER-2 at 1:500 (Upstate Biotechnology, Lake Placid, NY); p27 polyclonal at 1:250 and h-actin monoclonal at 1:5,000 (Santa Cruz Biotechnology).

Techniques: Western Blot, Immunoprecipitation, Staining, Liquid Chromatography with Mass Spectroscopy, Membrane, Control

Journal: Cancers

Article Title: Favorable Conditions for the Detection of EGFR T790M Mutation Using Plasma Sample in Patients with Non-Small-Cell Lung Cancer

doi: 10.3390/cancers15051445

Figure Lengend Snippet: Baseline characteristics of study subjects.

Article Snippet: In liquid biopsies, two methods were used, either an EGFR Mutation Detection Kit (PNA clampTM; Panagene, Daejeon, Republic of Korea) or a Cobas EGFR Mutation Test v2 (cobas ® ; Roche Molecular Systems, Pleasanton, CA, USA), on the plasma sample.

Techniques: Clinical Proteomics, Mutagenesis

Journal: Cancers

Article Title: Favorable Conditions for the Detection of EGFR T790M Mutation Using Plasma Sample in Patients with Non-Small-Cell Lung Cancer

doi: 10.3390/cancers15051445

Figure Lengend Snippet: EGFR mutation status on secondary biopsy.

Article Snippet: In liquid biopsies, two methods were used, either an EGFR Mutation Detection Kit (PNA clampTM; Panagene, Daejeon, Republic of Korea) or a Cobas EGFR Mutation Test v2 (cobas ® ; Roche Molecular Systems, Pleasanton, CA, USA), on the plasma sample.

Techniques: Mutagenesis, Clinical Proteomics

Journal: Cancers

Article Title: Favorable Conditions for the Detection of EGFR T790M Mutation Using Plasma Sample in Patients with Non-Small-Cell Lung Cancer

doi: 10.3390/cancers15051445

Figure Lengend Snippet: TNM, clinical stage, and the number of metastatic organs at the time of re-biopsy for the detection of EGFR T790M mutation *.

Article Snippet: In liquid biopsies, two methods were used, either an EGFR Mutation Detection Kit (PNA clampTM; Panagene, Daejeon, Republic of Korea) or a Cobas EGFR Mutation Test v2 (cobas ® ; Roche Molecular Systems, Pleasanton, CA, USA), on the plasma sample.

Techniques: Mutagenesis, Clinical Proteomics

Journal: Molecular Cancer

Article Title: Metabolism of the EGFR tyrosin kinase inhibitor gefitinib by cytochrome P450 1A1 enzyme in EGFR-wild type non small cell lung cancer cell lines

doi: 10.1186/1476-4598-10-143

Figure Lengend Snippet: Intracellular content of gefitinib in NSCLC cell lines and its effect on EGFR autophosphorylation . A) Time course of 0.1 μM [ 3 H]gefitinib accumulation in H322 (●) and H1299 (○) cell lines. Each point represents the mean ± SD of four independent determinations. B) H322 cells were incubated for 0.5 h and 24 h with the indicated extracellular concentrations of gefitinib before stimulation with 0.1 μg/ml EGF for 5 min. Western blot analysis was performed by using monoclonal antibodies directed to p-EGFR(p-Tyr1068) and to EGFR. The immunoreactive spots were quantified by densitometric analysis, ratios of phosphotyrosine/total EGFR were calculated and values are expressed as percentage of inhibition versus control. The experiment, repeated three times, yielded similar results. C) H322 cells were incubated with 0.1 μM gefitinib for 0.5 h or 24 h and then the initial rate (5 min) of 0.1 μM [ 3 H]gefitinib uptake was measured. Each bar represents the mean ± SD of four independent determinations. D) H322 cells were exposed to 0.1 μM [ 3 H]gefitinib for 0.5 h and 24 h, in the absence or in the presence of 10 μM Fumetrimorgin C (F) or PSC833 (P) and then intracellular gefitinib content was determined. Values given are the means (± SD) of three independent determinations (***P < 0.001).

Article Snippet: Anti-EGFR, anti-phospho-EGFR (tyr1068), anti-phospho-p44/42 MAPK, anti-p44/42 MAPK, anti-phospho-AKT (Ser473), anti-AKT and anti-actin were from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Incubation, Western Blot, Bioprocessing, Inhibition, Control

Journal: Molecular Cancer

Article Title: Metabolism of the EGFR tyrosin kinase inhibitor gefitinib by cytochrome P450 1A1 enzyme in EGFR-wild type non small cell lung cancer cell lines

doi: 10.1186/1476-4598-10-143

Figure Lengend Snippet: Production and biological activity of gefitinib metabolites . Cells were incubated with 0.1 μM gefitinib for 0.5, 6 or 24 h and then the M1 content was determined both in the intracellular (A) and extracellular (B) compartment, through LC-MS/MS analysis. M1 levels calculated for each cell line were expressed as pmol/mg of protein (intracellular) or μM (extracellular). Values given are the means (± SD) of three independent determinations C) H322 cells were exposed for 72 h to different concentrations of gefitinib or its metabolites (ranging from 0.05 to 10 μM) and then cell growth was assessed using crystal violet staining. Data are expressed as percent inhibition of cell proliferation versus control cells. The mean values of three independent measurements (± SD) are shown. D) H322 cells were incubated for 0.5 h with the indicated concentrations of gefitinib and metabolites, before stimulation with 0.1 μg/ml EGF for 5 min. Western blot analysis was performed by using monoclonal antibodies directed to p-EGFR(p-Tyr1068) and to EGFR. The immunoreactive spots at each point were quantified by densitometric analysis, ratios of phosphotyrosine/total EGFR were calculated and values expressed as percentage of inhibition versus control. The experiment, repeated three times, yielded similar results. E) H1299 cells were exposed for 72 h to different concentrations of gefitinib or its metabolites (ranging from 1 to 30 μM) and then cell growth was assessed using crystal violet staining. Data are expressed as percent inhibition of cell proliferation versus control cells. The mean values of three independent measurements (± SD) are shown.

Article Snippet: Anti-EGFR, anti-phospho-EGFR (tyr1068), anti-phospho-p44/42 MAPK, anti-p44/42 MAPK, anti-phospho-AKT (Ser473), anti-AKT and anti-actin were from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Activity Assay, Incubation, Liquid Chromatography with Mass Spectroscopy, Staining, Inhibition, Control, Western Blot, Bioprocessing

Journal: Molecular Cancer

Article Title: Metabolism of the EGFR tyrosin kinase inhibitor gefitinib by cytochrome P450 1A1 enzyme in EGFR-wild type non small cell lung cancer cell lines

doi: 10.1186/1476-4598-10-143

Figure Lengend Snippet: Effects of CYP1A1 inhibition on intracellular level of gefitinib, EGFR autophosphorylation and inhibition of cell growth . A) Calu-3 cells were incubated with 0.1 μM [ 3 H]gefitinib for 0.5, 24, 48 or 72 h in the absence or in the presence of 10 μM α-NAP. Values given of gefitinib content, expressed as pmol/mg of protein, are the means (± SD) of four independent determinations. α-NAP was renewed after 36 h of gefitinib treatment. (***P < 0.001). B) Calu-3 cells were treated with 0.1 μM gefitinib for 24 h in the absence or in the presence of 10 μM α-NAP, then the conditioned media (CM) were collected and extracts were prepared from H322 cells exposed for 2 h to CM. The effect of CM on EGF (0.1 μg/ml for 5 min)-induced EGFR autophosphorylation was examined by Western blotting using monoclonal antibodies directed against pEGFR(Tyr1068) and actin. The experiment, repeated twice, yielded similar results. Line 1: CM from control cells; line 2: CM from gefitinib treated cells; line 3: CM from α-NAP treated cells; line 4: CM from α-NAP and gefitinib treated cells. C) H322 and Calu-3 cells were incubated for 48 h with 0.1 μM gefitinib in the absence or in the presence of 10 μM α-NAP. Before protein extraction cells were stimulated with 0.1 μg/ml EGF for 5 min. Western blot analysis was performed by using monoclonal antibodies directed against pEGFR(Tyr1068), EGFR, p-p44/42 MAPK, p44/42 MAPK, pAKT (Ser473), AKT. The experiment, repeated three times, yielded similar results. α-NAP was renewed after 24 h of gefitinib treatment. D) Calu-3, (E) H322, (F) H292 were exposed for 72 h to different concentrations of gefitinib in the absence or in the presence of 10 μM α-NAP. Cell growth was assessed using crystal violet staining as described in Materials and Methods. Data are expressed as percent inhibition of cell proliferation versus control cells. The mean values of three independent measurements (± SD) are shown. α-NAP was renewed after 36 h of gefitinib treatment (*P < 0.05; **P < 0.01; ***P < 0.001).

Article Snippet: Anti-EGFR, anti-phospho-EGFR (tyr1068), anti-phospho-p44/42 MAPK, anti-p44/42 MAPK, anti-phospho-AKT (Ser473), anti-AKT and anti-actin were from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Inhibition, Incubation, Western Blot, Bioprocessing, Control, Protein Extraction, Staining

Journal: The Journal of Biological Chemistry

Article Title: Covalent N -arylation by the pollutant 1,2-naphthoquinone activates the EGF receptor

doi: 10.1016/j.jbc.2021.100524

Figure Lengend Snippet: Activation of Akt by 1,2-NQ via the PI3K–phosphoinositide-dependent protein kinase-1 pathway in A549 cells. A and B , cells were incubated with serum-free medium for 24 h at 37 °C and exposed to the indicated concentrations of 1,2-NQ for 15 min. The lysates were analyzed by Western blotting with anti-Akt or anti-phosphorylated Akt antibodies. C – E , cells were treated with the indicated concentrations of wortmannin ( C ), OSU-03012 ( D ), or BX-795 ( E ) for 10 min, 4 h, and 12 h, respectively, and were then stimulated with 20 μM 1,2-NQ for 15 min. F , cells were cultured with serum-free medium for 24 h prior to treatment with indicated concentrations of 1,2-NQ. G – I , cells were cultured with serum-free medium for 24 h prior to treatment with 20 μM 1,2-NQ, 1,2-DDN ( G ), or insulin ( H and I ) for 15 min. The levels of phosphorylated EGFR, total EGFR, phosphorylated IGF-1R, total IGF-1R, phosphorylated insulin receptor, and total insulin receptor were evaluated by Western blotting. J , cells were treated with the indicated concentrations of tyrphostin A25 for 12 h before stimulation with 20 μM 1,2-NQ. The cell lysates were then analyzed by Western blotting. Statistical analysis was carried out by one-way ANOVA with Bonferroni’s multiple comparisons test. All data are expressed as the mean ± SEM values. n = 3, ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 versus control. 1,2-DDN, 1,2-dihydronaphthalene; 1,2-NQ, 1,2-naphthoquinone; Akt, protein kinase B; EGFR, epidermal growth factor receptor; IGF-1R, insulin-like growth factor 1 receptor.

Article Snippet: Recombinant human EGFR (hEGFR; Sino Biological, Inc; 5 μM) was incubated with dimethyl sulfoxide as a control or 25 μM 1,2-NQ for 15 min at 25 °C in 50 mM ammonium bicarbonate.

Techniques: Activation Assay, Incubation, Western Blot, Cell Culture, Control

Journal: The Journal of Biological Chemistry

Article Title: Covalent N -arylation by the pollutant 1,2-naphthoquinone activates the EGF receptor

doi: 10.1016/j.jbc.2021.100524

Figure Lengend Snippet: 1,2-NQ directly induces EGFR dimerization and tyrosine phosphorylation. A and B , A549 cells were incubated with serum-free medium for 24 h and were then exposed to 10 μM 1,2-NQ or 10 ng/ml EGF for 10 min. Cells were pretreated with cetuximab ( A ) or panitumumab ( B ) for 3 h before stimulation with 1,2-NQ or EGF. C , human embryonic kidney 293T cells were transiently transfected with pIDT-SMART (C-TSC) human EGFR-FLAG. Cells were incubated with serum-free medium for 18 h and were then exposed to 10 μM 1,2-NQ for 10 min. Cells were pretreated with cetuximab for 3 h before stimulation with 1,2-NQ. Immunoprecipitation was performed under nonreducing conditions for the detection of EGFR dimerization ( upper ) and under reducing conditions for input analysis ( lower ). Western blotting was conducted with anti-1,2-NQ or anti-FLAG antibodies. D , quantification of 1,2-NQ–induced EGFR dimerization in A549 cells. The relative level of EGFR dimers was normalized to the input EGFR level and shown as a fold change compared with the control. Statistical analysis was carried out by one-way ANOVA with Bonferroni's multiple comparisons test. All data are expressed as the mean ± SEM values. n = 3, ∗ p < 0.05, and ∗∗ p < 0.01 versus control. E , A549 cells were cultured with serum-free medium for 24 h before exposure to 10 μM 1,2-NQ for 10 min. Cell lysates were analyzed by immunoprecipitation using an anti-EGFR antibody and by Western blotting with anti-1,2-NQ or anti-EGFR antibodies. F , A549 cells were incubated with serum-free medium for 24 h and were then exposed to 20 μM 1,2-NQ, 1 ng/ml EGF, or 1 ng/ml NRG-1 for 10 min. 1,2-NQ, 1,2-naphthoquinone; Akt, protein kinase B; DMSO, dimethyl sulfoxide; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; HER, human epidermal growth factor receptor; NRG-1, neuregulin-1.

Article Snippet: Recombinant human EGFR (hEGFR; Sino Biological, Inc; 5 μM) was incubated with dimethyl sulfoxide as a control or 25 μM 1,2-NQ for 15 min at 25 °C in 50 mM ammonium bicarbonate.

Techniques: Incubation, Transfection, Immunoprecipitation, Western Blot, Control, Cell Culture

Journal: The Journal of Biological Chemistry

Article Title: Covalent N -arylation by the pollutant 1,2-naphthoquinone activates the EGF receptor

doi: 10.1016/j.jbc.2021.100524

Figure Lengend Snippet: 1,2-NQ forms N -arylation with EGFR at Lys80 and induces its phosphorylation. A , identification of 1,2-NQ–binding sites in recombinant extracellular EGFR by ultra–high-performance liquid chromatography–tandem mass spectrometry analysis. Recombinant EGFR (5 μM) was incubated with 25 μM 1,2-NQ for 15 min at 25 °C in 50 mM ammonium bicarbonate. Trypsin-digested peptides were analyzed by ultra–high-performance liquid chromatography–tandem mass spectrometry. The mass data are shown in Fig. S4 . B and C , human embryonic kidney 293T cells were transiently transfected with WT, K80A, or K133A human EGFR. Cells were incubated with serum-free medium for 18 h after 6 h of transfection and were then stimulated with 10 μM 1,2-NQ for 10 min. The relative level of phosphorylated EGFR was normalized to the level of total EGFR. Statistical analysis was carried out by one-way ANOVA with Bonferroni's multiple comparisons test. All data are expressed as the mean ± SEM values. n = 3, ∗ p < 0.05, and ∗∗ p < 0.01 versus control. 1,2-NQ, 1,2-naphthoquinone; EGFR, epidermal growth factor receptor; ns, not significant.

Article Snippet: Recombinant human EGFR (hEGFR; Sino Biological, Inc; 5 μM) was incubated with dimethyl sulfoxide as a control or 25 μM 1,2-NQ for 15 min at 25 °C in 50 mM ammonium bicarbonate.

Techniques: Binding Assay, Recombinant, High Performance Liquid Chromatography, Mass Spectrometry, Incubation, Transfection, Control

Journal: The Journal of Biological Chemistry

Article Title: Covalent N -arylation by the pollutant 1,2-naphthoquinone activates the EGF receptor

doi: 10.1016/j.jbc.2021.100524

Figure Lengend Snippet: 1,2-NQ induces EGFR internalization. A , cells were incubated with serum-free medium for 3 h and were then stimulated with 10 μM 1,2-NQ for the indicated time. Immunofluorescence staining was performed to visualize EGFR internalization. The scale bar represents 50 μm. B , cells were pretreated with cetuximab in serum-free medium for 3 h and were then exposed to 1,2-NQ for 10 min. Immunofluorescence staining was performed. The scale bar represents 50 μm. C , quantification of EGFR internalization by exposure to 1,2-NQ or EGF for the indicated time. A549 cells were cultured in serum-free medium for 3 h and were then exposed to 1,2-NQ or EGF. 1,2-NQ-treated cells were pretreated with cetuximab in serum-free medium for 3 h. Statistical analysis was carried out by two-way ANOVA with Bonferroni’s multiple comparisons test. The data are expressed as the mean ± SEM values. n = 3, ∗ p < 0.05 versus control. D , cells were cultured in serum-free medium for 3 h and were then stimulated with 10 μM 1,2-NQ for the indicated time. Immunofluorescence staining was conducted to visualize the colocalization of EGFR and 1,2-NQ. 1,2-NQ, 1,2-naphthoquinone; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor.

Article Snippet: Recombinant human EGFR (hEGFR; Sino Biological, Inc; 5 μM) was incubated with dimethyl sulfoxide as a control or 25 μM 1,2-NQ for 15 min at 25 °C in 50 mM ammonium bicarbonate.

Techniques: Incubation, Immunofluorescence, Staining, Cell Culture, Control