Journal: Cellular signalling

Article Title: Features of the reversible sensitivity-resistance transition in PI3K/PTEN/AKT signalling network after HER2 inhibition.

doi: 10.1016/j.cellsig.2011.09.030

Figure Lengend Snippet: Fig. 2. Dose response and sensitivity properties of the PI3K/PTEN/AKT signalling network. (A) pHER2 dose dependence for heregulin-β (HRG) concentration (solid line), pAKT dose dependence for HRG in the absence (dashed line) and presence of PTEN inhibitor, 50 nM bpV(pic) (dotted line). (B) pHER2 and pAKT dose dependencies for pertuzumab (solid and dashed lines, respectively) at 95 nM and 1 μM of HER2 concentration (thick and thin lines, respectively). Dotted line — pAKT dose dependence at three-fold increase in activity of CK2/GSK3β reaction of PTEN phosphorylation. Points on thick lines — experimental data (see Fig. 3 in [33]). (C) pHER2 and pAKT dose dependencies for HER2 concentration in the absence (thick solid and dashed lines, respectively) and presence of 100 nM pertuzumab (thin solid and dashed lines, respectively). (D) The dependence of sensitivities of the whole signalling network, SSN (solid line), receptor subsystem SRSS (dotted line), and signalling transaction subsystem SSTS (dashed line) on pertuzumab concentration. (E) Western blot analysis of the dose dependence of pHER2 (left) and pAKT (right) to HRG concentration (0 nM (control), 0.01 nM, 0.1 nM, 1 nM and 10 nM) at 5 and 30 min.

Article Snippet: Cells were treatedwith UCN-01 (protein kinase inhibitor; Calbiochem#539644; final concentration of 1 μM), LY294002 (PI3 kinase inhibitor; Calbiochem#440204; final concentration 20 μM), pertuzumab (HER2 inhibitor; final concentration 100 nM) and stimulation by heregulin (R&D Systems; 396-HB-CF) was at final concentration of 1 nM.

Techniques: Concentration Assay, Activity Assay, Phospho-proteomics, Western Blot, Control

Journal: Cellular signalling

Article Title: Features of the reversible sensitivity-resistance transition in PI3K/PTEN/AKT signalling network after HER2 inhibition.

doi: 10.1016/j.cellsig.2011.09.030

Figure Lengend Snippet: Fig. 6. (A) Western blot analysis of the inhibition effect on pAKT of varying concentrations of PTEN inhibitor, bpV(pic) (5 nM, 10 nM, 25 nM, 50 nM, 100 nM) at 1 nM heregulin, HRG in PE04 cells. (B) Theoretical pAKT dose dependence on PTEN concentration at saturated HRG signal (thin line) and at HER2 inhibition by pertuzumab (thick line). Squares — ex- perimental data on the dependence of pAKT concentration on PTEN expression level in 13 ovarian cancer lines (in relative units). Circles — experimental data on the dependence of pAKT concentration on PTEN expression for basal-like breast carcinoma (in relative units) [28]. (C) Experimental data (mean±S.D., n=3) on the effects of combinations of PDK1 in- hibition by 7.5 μM UCN-01, HER2 inhibition by 100 nM pertuzumab (2C4) and PTEN inhi- bition by 50 μM bpV(pic).

Article Snippet: Cells were treatedwith UCN-01 (protein kinase inhibitor; Calbiochem#539644; final concentration of 1 μM), LY294002 (PI3 kinase inhibitor; Calbiochem#440204; final concentration 20 μM), pertuzumab (HER2 inhibitor; final concentration 100 nM) and stimulation by heregulin (R&D Systems; 396-HB-CF) was at final concentration of 1 nM.

Techniques: Western Blot, Inhibition, Concentration Assay, Expressing

Journal: Cellular signalling

Article Title: A novel epidermal growth factor receptor-signaling platform and its targeted translation in pancreatic cancer.

doi: 10.1016/j.cellsig.2013.08.008

Figure Lengend Snippet: Fig. 2. (A) Tamiflu and anti-Neu1 neutralizing antibody inhibit EGF induced EGFR phosphorylation (pEGFR) in 3T3–hEGFR cells. Cells were grown overnight on glass coverslips in a 24-well tissue culture plate at 37 °C for 24 h or until they reached ~70% confluence. Cells were stimulated with 30 ng/mL EGF for 5 min, pretreated with 200 μM Tamiflu for 30 min followed by 30 ng/mL EGF for 5 min, or pretreated with 100 μg/mL anti-Neu1, -2, -3, or -4 neutralizing antibodies for 30 min followed by 30 ng/mL EGF for 5 min. Cells were left untreated as no ligand controls. Cells were fixed with 4% paraformaldehyde, permeabilized with Triton X, and blocked with 4% bovine serum albumin (BSA) in 0.1% Tween–Tris buffered saline (TBS) for 20 min on ice. Cells were immunostained with rabbit anti-human pEGFR for 60 min at 37 °C, followed by AlexaFluor 594 goat anti-rabbit secondary antibody for 60 min at 37 °C. Control group had only secondary antibodies with no other treatment. Stained cells were visualized by epi-fluorescence microscopy with a 40× objective. Quantitative analysis was done by assessing the density of cell staining corrected for background in each panel using Corel Photo Paint 8.0 software. Each bar in the figure represents the mean corrected density of culture cell staining ± SEM for equal cell density (5 × 105 cells) within the respective images. The data are a representation of one out of three independent experiments showing similar results. (B) Western blot analyses of Tamiflu and anti-Neu1 neutralizing antibody inhibition of EGF-induced pEGFR in 3T3–hEGFR cell lysates. Cell were treated 30 ng/mL EGF for 5 min or pretreated with either 400 μM Tamiflu or 100 μg/mL anti-Neu1 neutralizing antibody for 30 min or left untreated as control (media). Cells were pelleted, lysed in lysis buffer and the cell lysates were resolved by SDS-PAGE. The blot was probed with 0.14 μg/mL rabbit anti-human pEGFR antibody overnight at 4 °C followed by 40 ng/mL horse radish peroxidase-labeled goat anti- rabbit antibody for 75 min at 20 °C and Western Lightning Chemiluminescence Reagent Plus for 5 min. NIH3T3 cells served as EGFR negative control. After development, blots were stripped and re-probed with rabbit anti-human pan-EGFR as a loading control. The data are a representation of one out of three independent experiments showing similar results. (C) A Western blot was performed as described in (B) above, but 3T3–hEGFR cells were stimulated with 30 ng/mL EGF for 10 min. (D) Immunoprecipitation of EGFR and Western blot analyses of biotinylated cell surface of 3T3–hEGFR cells in the presence of Tamiflu, anti-Neu1 neutralizing antibody and specific MMP-9 inhibitor. Cells were left untreated (control), stimulated with 30 ng/mL EGF for 5 min, or pretreated with 400 μM Tamiflu, 100 μg/mL anti-Neu1 neutralizing antibody or 50 μg/mL MMP-9 inhibitor for 30 min followed by 30 ng/mL EGF stimulation for 5 min. Cells were biotinylated with NHS-SS-biotin on ice for 30 min, extensively washed, pelleted and lysed in lysis buffer. The EGFR in the cell lysates was immunoprecipitated with 1 μg of goat anti-EGFR antibody overnight at 4 °C. Immunocomplexes were isolated using protein G magnetic beads, resolved by SDS-PAGE and the blot probed with streptavidin–HRP followed by Western Lightning Chemiluminescence Reagent Plus. NIH3T3 and 3T3–hEGFR cells that were not immunoprecipitated (no IP Ab) served as negative controls. The data are a representation of one out of two independent experiments showing similar results. (E) Maackia amurensis lectin 2 (MAL-2) dose-dependently inhibits EGF-induced pEGFR in human skin epidermoid carcinoma A431 cell line. A431 cells were starved in serum free media for 24 h. The cells were pretreated with MAL-2, Sambucus nigra lectin (SNA), peanut agglutinin (PNA) and succinylated wheat germ agglutinin (sWGA) lectins at indicated doses for 30 min followed by 30 ng/mL EGF for 5 min or left unstimulated as control. Cells were fixed with 4% paraformaldehyde, permeabilized with Triton X100 and immunostained with mouse anti-pEGFR followed by AlexaFluor 488 rabbit anti-mouse IgG. Stained cells were visualized by epi- fluorescence microscopy using a 40× objective. Quantitative analysis was done by assessing the density of cell staining corrected for background in each panel using Corel Photo Paint 8.0 software. Each bar in the graphs represents the mean corrected density of staining ± S.E. (error bars, n = 4) for equal cell density (5 × 105 cells) within the respective images. Results were compared by a one-way ANOVA at 95% confidence using Bonferroni's multiple comparison test. The data are a representation of one of five independent experiments showing similar results.

Article Snippet: Preclinical molecular-targeting studies focused on inhibiting Neu1 as the key central enzyme within this novel EGFR signaling paradigm provide the proof-of-evidence for an effective Tamiflu monotherapy in the treatment of human pancreatic cancer growth and metastatic spread in heterotopic xenograft of tumors growing in RAG2−/−xCγ−/− doublemutant mice.

Techniques: Phospho-proteomics, Saline, Control, Staining, Microscopy, Software, Western Blot, Inhibition, Lysis, SDS Page, Labeling, Negative Control, Immunoprecipitation, Isolation, Magnetic Beads, Comparison

Journal: Cellular signalling

Article Title: A novel epidermal growth factor receptor-signaling platform and its targeted translation in pancreatic cancer.

doi: 10.1016/j.cellsig.2013.08.008

Figure Lengend Snippet: Fig. 4. (A) Neu1 co-immunoprecipitates with EGFR. 3T3–hEGFR cells were treated with 30 ng/mL EGF for 5 min, or pretreated with 400 μM Tamiflu for 30 min followed by 30 ng/mL EGF for 5 min or left untreated as control (media). Cells were pelleted, lysed in lysis buffer and the protein lysates were immunoprecipitated with 1 μg of goat anti-EGFR antibody overnight at 4 °C. Immunocomplexes were isolated using protein G magnetic beads, resolved by SDS-PAGE and the blot probed with rabbit anti-Neu1 antibody overnight at 4 °C followed by HRP- conjugated goat anti-rabbit HRP-conjugated secondary antibody for 75 min at 20 °C and Western Lightning Chemiluminescence Reagent Plus. NIH3T3 and 3T3–hEGFR cells not immunoprecipitated (no IP Ab) served as negative controls. The data are a representation of one out of four independent experiments showing similar results. (B) Neu-2 and -3 do not co-immunoprecipitate with EGFR. 3T3–hEGFR cells were used as described in (A). (C) Western blot of unstimulated 3T3–hEGFR cells was run simultaneously with the immunoprecipi- tation to detect levels of Neu-2, -3, and -4 proteins in the same cell lysates. The data are a representation of one out of three independent experiments showing similar results.

Article Snippet: Preclinical molecular-targeting studies focused on inhibiting Neu1 as the key central enzyme within this novel EGFR signaling paradigm provide the proof-of-evidence for an effective Tamiflu monotherapy in the treatment of human pancreatic cancer growth and metastatic spread in heterotopic xenograft of tumors growing in RAG2−/−xCγ−/− doublemutant mice.

Techniques: Control, Lysis, Immunoprecipitation, Isolation, Magnetic Beads, SDS Page, Western Blot

Journal: Oncogene

Article Title: Glioma-specific Domain IV EGFR cysteine mutations promote ligand-induced covalent receptor dimerization and display enhanced sensitivity to dacomitinib in vivo.

doi: 10.1038/onc.2014.106

Figure Lengend Snippet: Figure 1. Biochemical characterization of Domain IV mutants. (a) Representative flow cytometry histograms for cell-surface EGFR expression, as determined by mAb528 binding after viral transduction of U87MG cells with vectors expressing the indicated mutants and subsequent drug selection. No cell sorting was conducted on the Domain IV mutant cell lines. (b) Eu-pY assay for sensitive detection of basal phosphorylation. Each cell line was serum-starved in the presence of vehicle, 1 μM lapatinib or 40 μg/ml EGFR 501-Fc ligand trap overnight before the assay was conducted. Data is presented as the mean time-resolved fluorescence (TRF) obtained for each test group over quadruple-technical replicates ± standard error (s.e.). Experiments were repeated three times. (c) Eu-pY assay for determination of global phosphotyrosine status of receptor in response to titration of ligand. Each cell line was serum-starved and treated with titrated concentrations of EGF (first graph), TGF-α (second graph), heparin-bound EGF (third graph) or betacellulin (fourth graph). Basal phosphorylation TRF reads were subtracted from all readings and then readings graphed as a percentage TRF obtained for each ligand concentration compared with the maximum stimulus obtained at 10 or 30 nM± s.e. Experiments were repeated three times.

Article Snippet: The anti-C-terminal EGFR (clone 1005) rabbit polyclonal antibody, either alone or conjugated to agarose beads, was from Santa Cruz (Dallas, TX, USA).

Techniques: Cytometry, Expressing, Binding Assay, Transduction, Selection, FACS, Mutagenesis, Phospho-proteomics, Titration, Concentration Assay

Journal: Oncogene

Article Title: Glioma-specific Domain IV EGFR cysteine mutations promote ligand-induced covalent receptor dimerization and display enhanced sensitivity to dacomitinib in vivo.

doi: 10.1038/onc.2014.106

Figure Lengend Snippet: Figure 2. Ligand-induced covalent dimerization of Domain IV EGFR mutants. (a–c) Nonreducing and reducing western blot analyzes for total EGFR (top panels) and pY1173 EGFR (middle panels). Overlays of the EGFR (red) and pY1173 (green) signal are shown to determine the active species (bottom panels). Dimeric (D) and monomeric (M) species for both wtEGFR, mutant EGFR and EGFRvIII are depicted by arrows. (a) The control cell line (U87MG) and cell lines expressing wtEGFR and EGFRvIII, treated with vehicle or EGF. (b) Cell lines expressing the Domain IV mutants C620Y, C624F, C628Y and C636Y, treated with vehicle or EGF. (c) Nonreducing western blot analyzes for total EGFR (top panels) and pY1173 EGFR (middle panels) in cell lines expressing wtEGFR, C620Y and C624F treated with the indicated ligands. In all figures, a pan-actin loading control is included.

Article Snippet: The anti-C-terminal EGFR (clone 1005) rabbit polyclonal antibody, either alone or conjugated to agarose beads, was from Santa Cruz (Dallas, TX, USA).

Techniques: Western Blot, Mutagenesis, Control, Expressing

Journal: Oncogene

Article Title: Glioma-specific Domain IV EGFR cysteine mutations promote ligand-induced covalent receptor dimerization and display enhanced sensitivity to dacomitinib in vivo.

doi: 10.1038/onc.2014.106

Figure Lengend Snippet: Figure 3. Analysis of cell-surface species present on Domain IV mutant cell lines. (a) Cells expressing wtEGFR, C620Y or C624F were treated at 4 °C with vehicle or EGF and the cell surface subsequently biotinylated. Streptavidin immunoprecipitations were performed after which western blot analyzes were performed for total EGFR (top panel) or pY1173 EGFR (middle panel), with overlays of the total EGFR (red) and pY1173 (green) signal shown to determine the active species (bottom panel). Dimeric (D) and monomeric (M) species are depicted by arrows. (b) Reciprocal experiments involving cell- surface biotinylation followed by EGFR immunoprecipitation and then western blot analyzes for streptavidin binding, indicating the total EGFR cell-surface species present. Dimeric (D) and monomeric (M) species are depicted by arrows. (c) wtEGFR, C620Y and C624F cell lines were cultured with or without 2 μg/ml of swainsonine overnight before cell lysis and western blot analyzes for total EGFR using an ECD- (top panel) or C-terminal domain-specific antibodies (middle panel). Dimeric (D) and monomeric (M) species are depicted by arrows. A pan-actin control was included for loading (bottom panel).

Article Snippet: The anti-C-terminal EGFR (clone 1005) rabbit polyclonal antibody, either alone or conjugated to agarose beads, was from Santa Cruz (Dallas, TX, USA).

Techniques: Mutagenesis, Expressing, Western Blot, Immunoprecipitation, Binding Assay, Cell Culture, Lysis, Control

Journal: Oncogene

Article Title: Glioma-specific Domain IV EGFR cysteine mutations promote ligand-induced covalent receptor dimerization and display enhanced sensitivity to dacomitinib in vivo.

doi: 10.1038/onc.2014.106

Figure Lengend Snippet: Figure 4. Dacomitinib-induced cytoplasmic retention of receptor and loss of ligand binding. (a) Cells expressing wtEGFR, C620Y, C624F or EGFRvIII were treated overnight with vehicle or 1 μM of gefitinib, dacomitinib or lapatinib. The EGF-alone control tests were stimulated the next day and cell lysates probed by nonreducing western blot analyzes for total EGFR (top panels) or pY1173 EGFR (middle panels). An overlay of the total EGFR (red) and pY1173 (green) signal are shown to determine the active species (bottom panels). Dimeric (D) and monomeric (M) species are depicted by arrows. (b) Representative images for cell-surface EGFR immunofluorescence conducted following overnight treatment of cells expressing wtEGFR, C620Y, EGFRvIII or C16S with vehicle (top panels) or 1 μM dacomitinib (bottom panels). Blue, nucleus; green, EGFR. Scale bar, 20 μm. (c) Cell-surface flow cytometry for EGFR status, as determined by panitumumab binding. Cells were treated overnight with vehicle or 1 μM dacomitinib, harvested and fixed before staining with isotype-control antibody or panitumumab. Red, isotype staining; blue, panitumumab staining in vehicle-treated cells; green, panitumumab staining in dacomitinib-treated cells. (d) Eu-EGF binding to cells after overnight treatment with vehicle or 1 μM dacomitinib. TRF readings were obtained for quadruple-technical replicates and graphed as a mean percentage of the vehicle in each cell line ± s.e. * represents significant differences compared with the respective vehicle control. Experiments were repeated three times.

Article Snippet: The anti-C-terminal EGFR (clone 1005) rabbit polyclonal antibody, either alone or conjugated to agarose beads, was from Santa Cruz (Dallas, TX, USA).

Techniques: Ligand Binding Assay, Expressing, Control, Western Blot, Cytometry, Binding Assay, Staining

Journal: Oncogene

Article Title: IGFBP-3 methylation-derived deficiency mediates the resistance to cisplatin through the activation of the IGFIR/Akt pathway in non-small cell lung cancer.

doi: 10.1038/onc.2012.146

Figure Lengend Snippet: Figure 4. Differential activation of EGF and IGFI receptors in 41S and 41R cell lines. (a) The protein levels of pEGFR, EGFR, pIGFIR and IGFIR were measured by WB. Cells were seeded and 24 h later incubated in serum-depleted medium for 24 h, then treated with the indicated CDDP doses for 6 h or with EGF or IGFI for 30 min as control stimuli. Total cell protein (20 mg) was subjected to WB and the membranes were hybridized with antibodies against pEGFR, pIGFIR, EGFR and IGFIR. (b) The 41R cell line were transiently transfected with pCMV5 (|) or with pCMV5-IGFBP-3 vectors, then cells were treated as in a. (c and d) IGFIR and EGFR cellular localization analyzed by immunofluorescence in 41S and 41R cell lines. Cells were grown on coverslips, 24 h later cells were shifted into medium containing 0.5% fetal bovine serum for 16 h, then cells were treated or not with IGFI or EGF as positive controls for 30 min, subsequently the coverslips were fixed and incubated with antibodies against IGFIR or EGFR, and then with a secondary antibody conjugate (fluorescent dye alexa fluor 488, Invitrogen, Carlsbad, CA, USA). The immunofluorescence was visualized with a confocal microscope.

Article Snippet: Antibodies used were anti-AKT (BD Biosciences, San Jose, CA, USA), pAKT-Ser473, PTEN, EGFR, pEGFR-Tyr1068 (1H12, WB), pEGFR-Tyr1068 (D7A5, IHC; Cell Signaling, Danvers, MA, USA), anti-IGFIR, anti-pIGFIRTyr1161 (Santa Cruz Biotechnology, Heidelberg, Germany), anti-phospho AKT-pS473 (Dakopatts, Glostrup, Denmark) and anti-a-tubulin (SigmaAldrich).

Techniques: Activation Assay, Incubation, Control, Transfection, Microscopy

Journal: Oncogene

Article Title: IGFBP-3 methylation-derived deficiency mediates the resistance to cisplatin through the activation of the IGFIR/Akt pathway in non-small cell lung cancer.

doi: 10.1038/onc.2012.146

Figure Lengend Snippet: Figure 5. Correlation between IGFBP-3 expression and IGFIR/AKT activation in different human cancer cell lines. The cell lines H1299, PANC-1, H727 and HT29 were transiently transfected with 1.5 mg pCMV5 (|) or with pCMV5-IGFBP-3 (IGFBP-3) vectors and 24 h later treated with CDDP at indicated doses for 6 h. Then RNA and protein extracts were obtained in parallel experiments. (a) IGFBP-3 overexpression was confirmed by RT–PCR in all cell lines and GAPDH mRNA was co-amplified as a loading control. (b and c) Total cell protein (20 mg) was subjected to WB and then membranes were hybridized with antibodies against pIGFIR, IGFIR, pEGFR, EGFR, p-AKT, AKT and a-tubulin as a loading control.

Article Snippet: Antibodies used were anti-AKT (BD Biosciences, San Jose, CA, USA), pAKT-Ser473, PTEN, EGFR, pEGFR-Tyr1068 (1H12, WB), pEGFR-Tyr1068 (D7A5, IHC; Cell Signaling, Danvers, MA, USA), anti-IGFIR, anti-pIGFIRTyr1161 (Santa Cruz Biotechnology, Heidelberg, Germany), anti-phospho AKT-pS473 (Dakopatts, Glostrup, Denmark) and anti-a-tubulin (SigmaAldrich).

Techniques: Expressing, Activation Assay, Transfection, Over Expression, Reverse Transcription Polymerase Chain Reaction, Control

Journal: Oncogene

Article Title: IGFBP-3 methylation-derived deficiency mediates the resistance to cisplatin through the activation of the IGFIR/Akt pathway in non-small cell lung cancer.

doi: 10.1038/onc.2012.146

Figure Lengend Snippet: Figure 6. Combined use of EGFR, IGFIR and AKT protein phosphorylation together with IGFBP-3 methylation status predicts a potential response to CDDP for NSCLC. A total of 25 NSCLC tissue samples were analyzed for stain localization and intensity. Immunohistochemistry (IHCs) were performed using polyclonal antibody that specifically recognizes p-EGFR or pIGFIR or p-AKT. Same samples were previously used for the analysis of IGFBP-3 gene methylation status measured by MSP.16. (a) Example of IHC analysis in the examined tumor samples. Primary tumor 17 presents positive p-EGFR staining, without p-IGFIR and p-AKT staining, whereas patient number 19 presents positive p-IGFIR and p-AKT staining without significant p-EGFR stain. (b) Nomogram representing prior probability of positive-negative test (both of them with a 50% of chance for being positive or negative) and the post-test probability.

Article Snippet: Antibodies used were anti-AKT (BD Biosciences, San Jose, CA, USA), pAKT-Ser473, PTEN, EGFR, pEGFR-Tyr1068 (1H12, WB), pEGFR-Tyr1068 (D7A5, IHC; Cell Signaling, Danvers, MA, USA), anti-IGFIR, anti-pIGFIRTyr1161 (Santa Cruz Biotechnology, Heidelberg, Germany), anti-phospho AKT-pS473 (Dakopatts, Glostrup, Denmark) and anti-a-tubulin (SigmaAldrich).

Techniques: Phospho-proteomics, Methylation, Staining, Immunohistochemistry

Journal: Cancer science

Article Title: Activation of epidermal growth factor receptor signaling by the prostaglandin E(2) receptor EP4 pathway during gastric tumorigenesis.

doi: 10.1111/j.1349-7006.2011.01847.x

Figure Lengend Snippet: Fig. 1. Gene expression levels of epidermal growth factor receptor (EGFR) ligands, EGFR family members (a) and a disintegrin and metalloproteinases (ADAMs) (b) in the stomach of the respective models (mean ± SD) calculated from microarray results. Asterisks indicate P < 0.05 versus the wild-type level. (c) Fluorescence immunostaining for phosphorylated EGFR at Tyr845 (green) in the gastric mucosa of the indicated genotype mice. DAPI staining for nuclei is visualized in red. Bars indicate 100 lm.

Article Snippet: Antibody for phosphorylated EGFR (Tyr845) (cell signaling), Ki-67 (DakoCytomation, Carpenteria, CA, USA), or active b-catenin (Millipore, Billerica, MA, USA) was used as the primary antibody.

Techniques: Gene Expression, Microarray, Fluorescence, Immunostaining, Staining

Journal: Oncogene

Article Title: Protein tyrosine phosphatase PTPN3 inhibits lung cancer cell proliferation and migration by promoting EGFR endocytic degradation.

doi: 10.1038/onc.2014.312

Figure Lengend Snippet: Figure 3. PTPN3-mediated Eps15 dephosphorylation accelerates sorting of EGFR for lysosomal degradation. (a) H1975 cells were treated without or with EGF (100 ng/ml) for 5 min and immunostained with anti-PTPN3 (red) and anti-EGFR (green) antibodies. Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) in blue. The insets show a higher magnification of the area enclosed within the white box. Bar, 10 μm. (b) H1975 cells stably expressing HA-tagged PTPN3, FLAG-tagged Eps15-Y850F or an empty vector control were treated with EGF-Alexa 488 (100 ng/ml) for 1 h at 4 °C. Cells were then incubated at 37 °C for 5, 10 and 15 min for the internalization of EGF-Alexa 488. The rate of EGF internalization was determined by flow cytometry as described in the Materials and methods. Data represent the mean ± s.d. of three independent experiments. (c) H1975 cells stably expressing HA-tagged PTPN3, FLAG-tagged Eps15-Y850F or an empty vector control were treated with EGF-Alexa 488 (100 ng/ml) for 5 min. Ectopic expression of PTPN3 or Eps15-Y850F caused the colocalization of EGF-Alexa 488 and LAMP-2, compared with controls. Nuclei were stained with DAPI in blue. The insets show a higher magnification of the area enclosed within the white box. Bar, 10 μm. (d) Percentage colocalization of EGF-Alexa 488 with LAMP-2 in (c). Data are represented as mean ± s.d. of triplicates, with an average of 10 cells scored per experiment. ***Po0.001. (e, f) H1975 cells stably expressing HA-tagged PTPN3, FLAG-tagged Eps15- Y850F or an empty vector control were treated with 100 μM Bafilomycin A1 (Baf-A1) for 60 min, followed by incubation with 100 ng/ml EGF for the indicated times. Cell lysates were analyzed by immunoblotting with antibodies as indicated.

Article Snippet: Lentiviral production and infection were performed as previously described.57 Antibodies used for the study were: anti-pMAPK (Sigma, St Louis, MO, USA), anti-MAPK (Sigma), anti-LAMP-2 (Abcam, Cambridge, UK), anti-EEA1 (Cell Signaling, Danvers, MA, USA), anti-LAMP1 (Abcam), anti-Met (Abcam), anti-EGFR and anti-phospho EGFR antibodies (Cell Signaling), anti-fascin (Developmental Studies Hybridoma Bank, Iowa City, IA, USA).

Techniques: De-Phosphorylation Assay, Staining, Stable Transfection, Expressing, Plasmid Preparation, Control, Incubation, Cytometry, Western Blot

Journal: Oncogene

Article Title: Protein tyrosine phosphatase PTPN3 inhibits lung cancer cell proliferation and migration by promoting EGFR endocytic degradation.

doi: 10.1038/onc.2014.312

Figure Lengend Snippet: Figure 4. PTPN3-mediated Eps15 dephosphorylation promotes EGFR endocytotic trafficking through a non-clathrin pathway. (a–c) Lysates of H1975 cells expressing an empty vector control, PTPN3, or Eps15-Y850F were fractionated by sucrose density gradient centrifugation, and aliquots were immunoblotted with anti-EGFR, anti-clathrin and anti-caveolin-1 antibodies. Fractions 3–5 were enriched with caveolin-1 and denoted as lipid raft fractions, whereas fractions 8–10 were enriched with clathrin and denoted as non-lipid raft fractions. (d) Quantitative analysis for the relative distribution of EGFR in lipid raft fractions and non-lipid raft fractions. Data are represented as mean ± s.d. of triplicates. *P o 0.05. (e, f) H1975 cells stably expressing HA-tagged PTPN3, Eps15-Y850F or an empty vector control were treated with 100 μM MβCD for 60 min, followed by incubation with 100 ng/ml EGF for the indicated times. Cell lysates were analyzed by immunoblotting with antibodies as indicated.

Article Snippet: Lentiviral production and infection were performed as previously described.57 Antibodies used for the study were: anti-pMAPK (Sigma, St Louis, MO, USA), anti-MAPK (Sigma), anti-LAMP-2 (Abcam, Cambridge, UK), anti-EEA1 (Cell Signaling, Danvers, MA, USA), anti-LAMP1 (Abcam), anti-Met (Abcam), anti-EGFR and anti-phospho EGFR antibodies (Cell Signaling), anti-fascin (Developmental Studies Hybridoma Bank, Iowa City, IA, USA).

Techniques: De-Phosphorylation Assay, Expressing, Plasmid Preparation, Control, Gradient Centrifugation, Stable Transfection, Incubation, Western Blot