Article Title: NOTCH3 inactivation increases triple negative breast cancer sensitivity to gefitinib by promoting EGFR tyrosine dephosphorylation and its intracellular arrest
Figure Lengend Snippet: Rafts depletion induces endogenous EGFR-PTPH1 interaction, EGFR dephopshorylation, and its intracellular arrest in MDA-MB-468 TNBC cells. a Immunofluorescence assay (IF) was performed by using anti-EGFR (green) and anti-GM1 (red) antibodies to reveal the endogenous EGFR-rafts colocalization, shown in yellow (merge). Nuclei were DAPI labeled (blue). b Raft (R) and non-raft (NR) fractions derived from Methyl-β-cyclodextrin (MβCD)-treated and untreated cells were used for immunoblot assay with anti-pEGFR (Y1173) (indicated as pEGFR) and anti-EGFR antibodies, to test activated and total EGFR expression in rafts compartment, respectively. Anti-transferrin and anti-GM1 antibodies were used as a fraction markers. c Cells have been activated with EGF ligand for the times indicated, in the presence or absence of MβCD: the expression of phospho-EGFR at tyrosine 1173 and 1068 residues and total EGFR was determined in whole cell extracts by immunoblot analysis using the specific indicated antibodies. d – f MDA-MB-468 cells were treated with MβCD and stimulated with EGF for 60 min: control or anti-PTPH1 antibody immunoprecipitates were probed with anti-EGFR, to detect the EGFR-PTPH1 binding, and with the anti-PTPH1 antibody, to show PTPH1 immunoprecipitated protein levels. The inputs indicated in the panel shows 5% of each total lysate d . Relative EGFR extracellular expression (EGFR EC ) was evaluated by FACS e . IF assay was performed by using anti-EGFR (red) antibody to reveal the endogenous EGFR intracellular localization. Nuclei were DAPI labeled (blue). White arrows indicated peri-nuclear EGFR localization in EGF stimulated MβCD-treated cells ( f ). a , f Representative single plane confocal IF images captured using a × 60 oil objective. Scale bar: 10 μm. In both b and c , western blotting against the anti-β-actin was used as a loading control. All data are representative of at least three independent experiments, each in triplicate. Results shown in e are expressed as the means average deviations and P -values were calculated using Student’s T -test (i.e., ns, not significant P > 0.05, ** P ≤ 0.01)
Article Snippet: Cells were treated with the following compounds: 5 mM MβCD (Sigma-Aldrich, Catalog number C4555); 3 μM GEF (Iressa, Selleckem, Houston, TX, USA; Catalog number ZD1839), 100 nM EGF Ligand (EGF; Gibco, Life Technologies, Carlsbad, CA, USA; Catalog number PHG0315); 10 μM of GSI IX (DAPT) (Calbiochem, Darmstadt, Germany; Catalog number 565770).
Techniques: Multiple Displacement Amplification, Immunofluorescence, Labeling, Derivative Assay, Expressing, Binding Assay, Immunoprecipitation, FACS, Western Blot