Journal: Frontiers in Oncology

Article Title: Characterizing the secretome of EGFR mutant lung adenocarcinoma

doi: 10.3389/fonc.2023.1286821

Figure Lengend Snippet: Experiment overview, model expression validation, and GO enrichment analysis of identified proteins. (A) Schematic overview of model, where each cell line represents a stage of malignant transformation. HBEC GFP;p53wt represents the wild-type, untransformed state, HBEC cell lines expressing mutations in EGFR-driven transformation represent an intermediate pre-malignant stage, and EGFR mutant NSCLC cell lines PC-9, HCC4006, H1975, HCC4011, and H3255 represent the transformed, malignant state. (B) Western blot expression validation of HBEC stable cell lines expressing GFP, EGFR L858R, in combination with p53 CT. Cell lines were treated with 10µM Nutlin 3-a, an MDM2 inhibitor, for 24 hours to confirm mutant p53 expression. EGFR L858R basal expression was also confirmed. H1975 was used as a positive control. (C) Schematic overview of the secretome collection experiment. (D) Bar plots showing the top 20 GO enrichment terms sorted by adjusted p-value (p adj< 0.05, Benjamini-Hochberg adjustment) for biological processes, molecular functions, and cellular components, for proteins identified in 2 or more technical replicates (prior to statistical analysis).

Article Snippet: Lentiviral vector and overexpression plasmids used to construct overexpression constructs for EGFR L858R (Plasmids #82906, #17451) and GFP (Plasmid #17445) were obtained from Addgene.

Techniques: Expressing, Biomarker Discovery, Transformation Assay, Mutagenesis, Western Blot, Stable Transfection, Positive Control

Journal: International Journal of Nanomedicine

Article Title: Cetuximab-conjugated iron oxide nanoparticles for cancer imaging and therapy

doi: 10.2147/ijn.s80134

Figure Lengend Snippet: Figure 3 EGFRvIII-ECD-hFc binding determined by enzyme-linked immunosorbent assay. Notes: Soluble EGFRvIII-ECD-hFc was coated on enzyme-linked immunosorbent assay plates, and binding of cetuximab (A) or cetuximab-functionalized and non-functionalized iron oxide nanoparticles (B) was detected using a horseradish peroxidase-conjugated goat anti-human kappa light chain antibody. The concentrations tested ranged from 1.34×10-4 to 10 µg/mL for the anti-EGFR monoclonal antibody cetuximab and from 6.4×10-4 to 120 µg Fe/mL for cet-PEG-dexSPIONs and PEG-dexSPIONs. The EC50 was 0.0242 and 0.0646 µg/mL for cetuximab and cet-PEG-dexSPIONs, respectively. Each point represents the mean ± standard deviation of three determinations. Abbreviations: cet, cetuximab; dex, dextran; ECD, extracellular domain; EGFR, epithelial growth factor receptor; OD, optical density; PEG, polyethylene glycol; SPIONs, superparamagnetic iron oxide nanoparticles.

Article Snippet: The membranes were then incubated with primary antibodies against phospho-EGFR (Tyr1173; 1:2,000; Novus Biologicals Inc, Littleton, CO, USA) and β-actin (1:10,000; GeneTex Inc, Irvine, CA, USA), followed by horseradish peroxidaseconjugated goat anti-rabbit IgG (H + L) secondary antibodies (1:10,000; Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Nature Communications

Article Title: Intratumor heterogeneity of EGFR expression mediates targeted therapy resistance and formation of drug tolerant microenvironment

doi: 10.1038/s41467-024-55378-5

Figure Lengend Snippet: A Sorting scheme. Three EGFR -mutant cell lines were sequentially sorted for total EGFR using a viability marker. B EGFR expression in parental PC-9 cells before sequential sorting. IgG-488 was used as a negative control. C EGFR expression in PC-9 parental versus EGFR-low and EGFR-high cell lines after 6 rounds of sequential sorting. IgG-488 was used as a negative control. D EGFR expression in all three cell lines after 6 rounds of sorting. IgG-488 was used as a negative control. E – G EGFR mRNA expression in PC-9 / DFCI-284 / H1975, EGFR-high, and EGFR-low cell lines. N = 3 biologically independent experiments. H PC-9 EGFR-low and EGFR-high cells have differential expression on mutant EGFR. Western blot analysis showing total EGFR and EGFR E476-A750del expression. Hsp90 was used as a loading control. I PC-9 EGFR-low and EGFR-high cells have similar allelic frequencies. EGFR del19 / EGFR wt allelic frequency in PC-9 EGFR-low versus PC-9 EGFR-high cells. Allelic frequency was measured both in RNA and DNA level. N = 3 biologically independent experiments. J PC-9 EGFR-high cells are enriched with EGFR signaling compared to the EGFR-low cells. Gene-set enrichment analysis (GSEA). Three biological replicates. Data in ( E , F , G , I ) are presented as mean ± SD and analyzed by an unpaired student’s t- test.

Article Snippet: Mutant EGFR (pDONR233_EGFR_p.ELREA_746del, Addgene #82911) was cloned into pLEX_305_C’dTAG (kindly provided by Prof. Behnam Nabet ,) lentiviral plasmid by using Gateway cloning, obtaining mutEGFR-dTAG plasmid.

Techniques: Mutagenesis, Marker, Expressing, Negative Control, Western Blot, Control

Journal: Organoids

Article Title: Resistance to MAPK Pathway Inhibition in BRAF-V600E Mutant Colorectal Cancer Can Be Overcome with Insulin Receptor/Insulin-like Growth Factor-1 Receptor Inhibitors

doi: 10.3390/organoids4020014

Figure Lengend Snippet: Figure 2. EGFR inhibition induces compensatory activation of the insulin receptor (IR) and IGF-1 receptor (IGF1R). (a) The phosphorylation status of RTKs in afatinib-treated HUB040 organoids was assessed by Human phospho-RTK array. Representative dot images from the phospho-RTK array, arrows indicate the reference spots on the array blot. (b) Relative phospho-EGFR, phopho-IR, and phospho-IGF1R levels of HUB040 treated with afatinib. (c) Immunoblot analysis of HUB040 and P19bT organoids treated for 24 h with indicated drugs (1 µM each drug).

Article Snippet: Membranes were probed with antibodies directed against phospho-MEK1/2 (CST9121), MEK1/2 (CST9122), phospho-ERK1/2 (CST9101), ERK1/2 (CST4695), phospho-EGFR (CST2234), EGFR (CST2646), phospho-IGF1R/IR (CST3021), IGF1R (sc-81667), phospho-AKT (CST9271), AKT (CST9272), and phospho-S6 ribosomal protein (CST2211). β-actin (Novus Biologicals, Littleton, CO, USA, NB600-501) was used as an internal control.

Techniques: Inhibition, Activation Assay, Phospho-proteomics, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: The Impact of Inadequate Exposure to Epidermal Growth Factor Receptor–Tyrosine Kinase Inhibitors on the Development of Resistance in Non-Small-Cell Lung Cancer Cells

doi: 10.3390/ijms25094844

Figure Lengend Snippet: IC50 values for gefitinib in parental and gefitinib-resistant (GR) cell lines.

Article Snippet: EGFR-mutant (p.E746_A750del) NSCLC cell lines, HCC827 and PC9, were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and from the European Collection of Authenticated Cell Cultures (ECACC, Salisbury, UK), respectively.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: The Impact of Inadequate Exposure to Epidermal Growth Factor Receptor–Tyrosine Kinase Inhibitors on the Development of Resistance in Non-Small-Cell Lung Cancer Cells

doi: 10.3390/ijms25094844

Figure Lengend Snippet: Aberrant activation of EGFR signaling in GR cells. ( a , b ) Western blot analysis of EGFR expression and activation levels in HCC827 ( a ) and PC9 ( b ) parental and GR cells, in the absence or presence of gefitinib at the indicated concentrations. An anti-α-tubulin antibody was used for normalization. ( c , d ) Western blot analysis of expression and activation levels of ERK and AKT in HCC827 ( c ) and PC9 ( d ) parental and GR cells, in the absence or presence of gefitinib at the indicated concentrations. An anti-GAPDH antibody was used for normalization. Densitometric value ratios were calculated for phospho-ERK/total ERK (pERK/ERK) and phospho-AKT/total AKT (pAKT/AKT).

Article Snippet: EGFR-mutant (p.E746_A750del) NSCLC cell lines, HCC827 and PC9, were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and from the European Collection of Authenticated Cell Cultures (ECACC, Salisbury, UK), respectively.

Techniques: Activation Assay, Western Blot, Expressing

Journal: International Journal of Molecular Sciences

Article Title: The Impact of Inadequate Exposure to Epidermal Growth Factor Receptor–Tyrosine Kinase Inhibitors on the Development of Resistance in Non-Small-Cell Lung Cancer Cells

doi: 10.3390/ijms25094844

Figure Lengend Snippet: Evaluation of TGF-β1 expression in GR cells and its role in resistance and cell migration. ( a ) Immunofluorescence analysis of TGF-β1 expression (signaled in red) in HCC827 and PC9 parental and GR cell lines. Nuclei were stained with DAPI (signaled in blue). In the images, 20× magnification was used. ( b ) Immunoassay analysis of TGF-β1 levels in conditioned media from parental and GR cell lines. TGF-β1 levels were normalized for the cell number determined at the harvesting time and folds were calculated versus their respective parental cells. The values reported are the means from two independent experiments, each performed in duplicate. * p < 0.05 for comparison between parental versus GR cells (two-tailed Student’s t -test). ( c ) Cell proliferation assay on HCC827 parental and GR cells treated for 72 h with gefitinib (100 nM) and LY2109761 (LY, 5 µM), alone and in combination. *** p < 0.0005 for comparison between cells treated with gefitinib alone versus those treated with the drug combination (two-tailed Student’s t -test). ( d ) Analysis of wound-healing assays for HCC827 parental and GR-Low cells untreated and treated with LY (5 µM). *** p < 0.0005 for comparison between the treated versus untreated cells (two-tailed Student’s t -test).

Article Snippet: EGFR-mutant (p.E746_A750del) NSCLC cell lines, HCC827 and PC9, were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and from the European Collection of Authenticated Cell Cultures (ECACC, Salisbury, UK), respectively.

Techniques: Expressing, Migration, Immunofluorescence, Staining, Comparison, Two Tailed Test, Proliferation Assay