tyr845 phosphorylated egfr  (Cell Signaling Technology Inc)

Journal: Molecular Medicine Reports

Article Title: Propofol induces apoptosis and ameliorates 5-fluorouracil resistance in OSCC cells by reducing the expression and secretion of amphiregulin

doi: 10.3892/mmr.2021.12552

Figure Lengend Snippet: Propofol inhibits AREG expression in oral squamous cell carcinoma cells, which may reduce the activation of its related signaling pathways and lead to cell apoptosis. (A) mRNA and (B) protein expression levels of AREG were determined by reverse transcription-quantitative PCR (n=4-6) and western blotting (n=3), respectively. SAS and SCC9 cells were treated with propofol (10–160 µM) for 48 h. *P<0.05 vs. untreated control. (C) SAS and SCC9 cells were stimulated with or without rAREG (50 ng/ml) for 15 min. The activation of EGFR was determined using a human EGFR phosphorylation antibody array. (D) SAS and SCC9 cells were stimulated with different concentrations of rAREG (50–100 ng/ml) for 15 min and then the cell lysates were analyzed by western blotting using anti-p-EGFR (Tyr845) or anti-EGFR antibodies (n=3). (E) SAS cells were pretreated with rAREG (50 ng/ml) for 4 h and then stimulated with 40 µM propofol for 48 h (n=4). SCC9 cells were pretreated with rAREG (100 ng/ml) for 4 h and then stimulated with 80 µM propofol for 48 h (n=4). Cell viability was determined using an MTT assay (n=4). Data are presented as the mean ± SEM. *P<0.05 vs. Untreated control group; # P<0.05 vs. propofol. AREG, amphiregulin; rAREG, recombinant AREG; p, phosphorylated.

Article Snippet: The following primary antibodies were used: Cleaved PARP (1:2,000; cat. no. ab32064; Abcam), α-tubulin (1:10,000; cat. no. 05–829; EMD Millipore), AREG (1:200; cat. no. sc-74501; Santa Cruz Biotechnology, Inc.), phosphorylated (p)-EGFR (1:1,000; cat. no. 6963S; Cell Signaling Technology, Inc.), and EGFR (1: 200; cat. no. sc-53274; Santa Cruz Biotechnology, Inc.).

Techniques: Expressing, Activation Assay, Protein-Protein interactions, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Control, Phospho-proteomics, Ab Array, MTT Assay, Recombinant

Journal: Molecular Medicine Reports

Article Title: Propofol induces apoptosis and ameliorates 5-fluorouracil resistance in OSCC cells by reducing the expression and secretion of amphiregulin

doi: 10.3892/mmr.2021.12552

Figure Lengend Snippet: AREG upregulation is related to the development of 5-FU resistance, but propofol alleviates 5-FU resistance. (A) Cells were treated with 160 µM 5-FU for 48 h and then cell viability was determined by performing MTT assays. Data are presented as the mean ± SEM (n=3). *P<0.05 vs. SCC9. (B) AREG protein expression levels in 5-FU-resistant cell sub-lines and their parental cells were analyzed by western blotting with α-tubulin as the loading control (n=3). (C) Secreted levels of AREG in the cell culture media of SCC9 and SCC9-FUR160 cells were analyzed using western blotting (n=3). Ponceau S staining demonstrated that equivalent amounts of total protein were loaded on each lane. (D) Basal expression of p-EGFR and total EGFR in SCC9 and SCC9-FUR160 cells was evaluated by western blotting (n=3). (E) SCC9 cells were transiently transfected with pCMV3-AREG and then the protein expression level of AREG was analyzed by western blotting (n=3). (F) Cells were treated with increasing concentrations of propofol (0–160 µM) for 72 h. Effect of AREG overexpression on 5-FU-induced cytotoxicity was determined by performing MTT assays (n=4). *P<0.05 vs. pCMV. (G) Cells were treated with propofol (10 µM) for 24 h and then stimulated with 5-FU (40 µM) in the presence of 10 µM propofol for 48 h. Cell viability was then assessed by performing MTT assays (n=3). *P<0.05 vs. 5-FU treatment alone. AREG, amphiregulin; 5-FU, 5-fluorouracil; p, phosphorylated; FUR, 5-FU-resistant. F, 5-FU.

Article Snippet: The following primary antibodies were used: Cleaved PARP (1:2,000; cat. no. ab32064; Abcam), α-tubulin (1:10,000; cat. no. 05–829; EMD Millipore), AREG (1:200; cat. no. sc-74501; Santa Cruz Biotechnology, Inc.), phosphorylated (p)-EGFR (1:1,000; cat. no. 6963S; Cell Signaling Technology, Inc.), and EGFR (1: 200; cat. no. sc-53274; Santa Cruz Biotechnology, Inc.).

Techniques: Expressing, Western Blot, Control, Cell Culture, Staining, Transfection, Over Expression

Journal: bioRxiv

Article Title: Glioblastoma mutations impair ligand discrimination by EGFR

doi: 10.1101/2021.05.04.442654

Figure Lengend Snippet: a. Distribution of mutations in the extracellular region of EGFR found in GBM in the COSMIC database , with the most common non-cysteine mutations colored red and frequency (number of occurrences in COSMIC ) depicted by bar height. R198C and R228C are represented as red asterisks. Residue numbering corresponds to the mature form of EGFR, subtracting 24 for the signal sequence (R84 is R108 in pro-EGFR and A265 is A289). Below the graph is a schematic of the EGFR extracellular region domain structure. b. Cartoon of a symmetric dimer of the EGFR extracellular region (sEGFR) induced by EGF (black), with associated proliferative response in MCF7 cells . Individual domains are numbered. c. Cartoon of the asymmetric wild-type sEGFR dimer induced by EREG (magenta), with associated differentiative response in MCF7 cells . d. Structure of the EREG-induced asymmetric wild-type sEGFR dimer (PDBID 5WB7) . The left-hand molecule (grey) resembles sEGFR in symmetric ligand-bound sEGFR dimers, with a ligand-induced bend in domain II (see curve in domain II cartoon in left-hand side of c ). The right-hand molecule (light green) does not have the ligand-induced bend in domain II (see straight line in right-hand molecule in c ). Key residues mutated in GBM are colored cyan (left) or magenta (right), and are labelled in the inserts, as is F263. Note that the right-hand insert is rotated 180° about a vertical axis to allow comparison with L38/R84/A265 positions in the lefthand molecule.

Article Snippet: Chemiluminescence signals for phosphorylated EGFR (pY845: CST #2231) and ERK1/2 (pT202/pY204: CST #9106), were visualized and quantitated using a Kodak Image Station 440CF (Kodak Scientific).

Techniques: Residue, Sequencing, Comparison

Journal: bioRxiv

Article Title: Glioblastoma mutations impair ligand discrimination by EGFR

doi: 10.1101/2021.05.04.442654

Figure Lengend Snippet: a. IL-3-dependent Ba/F3 cells were stably transfected with human wild-type EGFR or with variants harboring an R84K or A265V mutation. Cells were either left untreated or were treated with IL-3 (2 ng/ml) for 72 h, after which a CyQuant Direct proliferation assay was used to detect the number of viable cells in each condition, and resulting fluorescence signals were normalized to that seen with untreated wild-type and shown as mean ± SD (n = 3 experiments). IL-3 treatment promotes robust proliferation in all cases (as positive control). As previously reported ( ; ; ), the mutated EGFRs promoted statistically significant increases in viable cell numbers compared with wild-type EGFR in the absence of ligand, but the effects were small at these expression levels – with the number of viable cells increased (compared with wild-type) by 1.31-fold (p = 0.0002) for R84K and 1.35-fold for A265V (p = 0.0003). p values are for unpaired two-tailed Student’s t-tests. b. Full-length human EGFR – wild-type or harboring an R84K, T239P or A265V mutation – was stably expressed in the engineered haploid eHAP cell line , which has negligible levels of endogenous EGFR (undetectable by Western blotting). Stably transfected cells were serum-starved overnight and either left unstimulated or stimulated with EGF (100 ng/ml) for the indicated times. Levels of phosphorylated EGFR (pY845: CST #2231) and ERK1/2 (pT202/pY204: CST #9106), were then detected by immunoblotting of total cell lysates, with blotting for Grb2 used as loading control ( ; ). Both EGFR phosphorylation and ERK phosphorylation are ligand-dependent in all cases, with no evidence for constitutive activation of the mutated receptors. c. Ba/F3 cells stably transfected with wild-type or mutated EGFR were sorted by flow cytometry to yield cell populations expressing similar levels of EGFR at their surface. Cells were either left untreated or were treated with similarly saturating levels of EGF (2 nM) or EREG (100 nM) for 72 hours. The CyQuant Direct proliferation assay was used to detect the number of viable cells in each condition. Fluorescence signals were normalized by signals from untreated cells and are shown as mean ± SD (n = 3 experiments). With equal receptor expression levels, A265V-mutated EGFR showed no significant differences compared with wild-type EGFR. However, R84K and A265V showed particularly large increases in EREG-induced proliferation compared to wild-type.

Article Snippet: Chemiluminescence signals for phosphorylated EGFR (pY845: CST #2231) and ERK1/2 (pT202/pY204: CST #9106), were visualized and quantitated using a Kodak Image Station 440CF (Kodak Scientific).

Techniques: Stable Transfection, Transfection, Mutagenesis, CyQUANT Assay, Proliferation Assay, Fluorescence, Positive Control, Expressing, Two Tailed Test, Western Blot, Control, Phospho-proteomics, Activation Assay, Flow Cytometry