Journal: Molecular Vision

Article Title: Glucosamine inhibits epidermal growth factor-induced proliferation and cell-cycle progression in retinal pigment epithelial cells

doi:

Figure Lengend Snippet: Effects of glucosamine (GlcN) on transactivation of epidermal growth factor receptor (EGFR). Human retinal pigment epithelial cell line (ARPE-19) cells were treated with 2.5 mM or 5.0 mM GlcN, or 30 mM glucose for 24 h, stimulated with 10 ng/ml EGF, and stained with PE-conjugated anti-total-EGFR antibody and Alexa-Fluor-647-conjugated anti-p-EGFR antibody (Y845). The levels of phosphorylated ( A ) and total EGFR ( B ) were analyzed by flow cytometry. The data are representative of at least three independent experiments. Cells cultured under serum-free conditions were used as the control. C : western blot analysis of ARPE-19 cells cultured to 80% confluence; treated with 2 μg/ml tunicamycin, 30 mM glucose, or 5 mM GlcN in serum-free medium for a further 24 h; then stimulated with EGF for 5 min.

Article Snippet: The following primary antibodies were used: phycoerythrin (PE)-conjugated anti-total-EGFR (cells were not permeabilized for total EGFR detection) and Alexa-Fluor-647-conjugated anti-p-EGFR (Y845; BD PharMingen).

Techniques: Staining, Flow Cytometry, Cell Culture, Western Blot

Journal: Oncotarget

Article Title: TRAIL-R2 promotes skeletal metastasis in a breast cancer xenograft mouse model

doi:

Figure Lengend Snippet: Control (Ctrl-shRNA) and TRAIL-R2 knockdown cells (R2-shRNA-1 or R2-shRNA-2) were analyzed in regard to their migration capacity towards SDF-1 in a trans-well assay (A) . Expression of TRAIL-R1, TRAIL-R2, CXCR4 and EGFR was assessed by flow cytometry on non-permeabilized and permeabilized cells (B) and percent of stained cells (C) , as well as staining intensities per cell (D) , relative to non-specific antibody controls, were quantified. Graphs represent average values ± SD. (A: n = 4, B-D: n = 3) (* p < 0.05, ** p < 0.01 *** p < 0.001).

Article Snippet: Briefly, 5 × 10 4 cells were seeded in 96-well plates and non-permeabilized or fixed and permeabilized (1% formalin, 30 min, 4 o C, washing, 0.1% Tween, 30 min, 4 o C, 2x washing) cells were incubated for 30 min at 4 o C with primary antibodies against CXCR4 (BD Bioscience, San Jose, CA, USA), EGFR (AnaSpec Inc, Fremont, CA, USA), TRAIL-R2 or TRAIL-R1 (HS201 and HS101 both kindly provided by Henning Walczak, Imperial College, London).

Techniques: shRNA, Migration, Expressing, Flow Cytometry, Staining

Journal: Oncotarget

Article Title: TRAIL-R2 promotes skeletal metastasis in a breast cancer xenograft mouse model

doi:

Figure Lengend Snippet: Expression of TRAIL-R2, CXCR4 and EGFR in control cells (pCR3.1) and cells overexpressing the long (R2-long) or short (R2-short) isoforms was assessed by flow cytometry in non-permeabilized and permeabilized cells and percent of stained cells (A) and staining intensities per cell (B) , relative to non-specific antibody controls, were quantified. (C) Cells were analyzed in regard to their migration capacity towards SDF-1 in a trans-well assay. Graphs represent average values ± SD. (A–B: n = 3, C: n = 4) (* p < 0.05, ** p < 0.01 *** p < 0.001).

Article Snippet: Briefly, 5 × 10 4 cells were seeded in 96-well plates and non-permeabilized or fixed and permeabilized (1% formalin, 30 min, 4 o C, washing, 0.1% Tween, 30 min, 4 o C, 2x washing) cells were incubated for 30 min at 4 o C with primary antibodies against CXCR4 (BD Bioscience, San Jose, CA, USA), EGFR (AnaSpec Inc, Fremont, CA, USA), TRAIL-R2 or TRAIL-R1 (HS201 and HS101 both kindly provided by Henning Walczak, Imperial College, London).

Techniques: Expressing, Flow Cytometry, Staining, Migration

Journal: Frontiers in Pharmacology

Article Title: Oxyresveratrol as a novel ferroptosis inducer exhibits anticancer activity against breast cancer via the EGFR/PI3K/AKT/GPX4 signalling axis

doi: 10.3389/fphar.2024.1527286

Figure Lengend Snippet: ORes induces ferroptosis in breast cancer cells via the EGFR/PI3K/AKT/GPX4 signalling axis. (A) The table indicates the top 10 connectivity scores between the input gene signature and the gene signatures of compounds in the LINCS L1000 touchstone dataset. (B) The table displays the top 10 scores between the input gene signature and the gene signatures of compounds in the DGDB dataset. (C) GSEA of HTS 2 results of Osimertinib dimesylate–treated MDA-MB-231 cells. (D) GSEA of HTS 2 results of AZ-5104–treated MDA-MB-231 cells. (E) Volcano plot of the DEGs in EGFR knockdown MDA-MB-231 cells, with red and blue dots indicating upregulated and downregulated genes, respectively. Differential gene screening criteria: |FoldChange|>1.3 and p- value < 0.05. (F) Representative Western blot results. (G) Quantification of western blots. Experiments were performed in triplicate, and data are presented as mean ± SD. *** p < 0.001; ns, no significance.

Article Snippet: The membranes were incubated overnight at 4°C with primary antibodies against GPX4 (Cell Signaling Technology, 59735, 1:1,000), phosphorylated EGFR (ZenBio, R26283, 1:1,000), EGFR (Selleck, A5858, 1:1,000), phosphorylated PI3K (ZenBio, 310164, 1:1,000), PI3K (ZenBio, 200900, 1:1,000), phosphorylated AKT (Cell Signaling Technology, 13038, 1:1,000), and AKT (Cell Signaling Technology, 4691, 1:1,000).

Techniques: Knockdown, Western Blot

Journal: PLoS ONE

Article Title: Identification of Human Fibroblast Cell Lines as a Feeder Layer for Human Corneal Epithelial Regeneration

doi: 10.1371/journal.pone.0038825

Figure Lengend Snippet: Corneal epithelial markers, including the differentiation markers, keratin 3 (K3), and connexin 43 (Cx43), as well as progenitor markers, EGFR, nuclear p63 and integrin β1, were expressed by HLE regenerated on human feeder of Hs68 fibroblasts; Hoechst 33342 was used for nuclear counterstaining.

Article Snippet: Immunofluorescent staining were performed with our previous methods , , , using primary antibodies against p63 , integrin β1, EGFR, K14, K3, connexin 43 (Calbiochem, Labvision, ICN Pharmaceuticals, Santa Cruz Biotechnology, Chemicon International, or Invitrogen, respectively), with Alexa Fluor 488 conjugated secondary antibodies (Invitrogen), and counter-staining with Hoechst 33342 (Sigma).

Techniques:

Journal: PLoS ONE

Article Title: Identification of Human Fibroblast Cell Lines as a Feeder Layer for Human Corneal Epithelial Regeneration

doi: 10.1371/journal.pone.0038825

Figure Lengend Snippet: Properties of human corneal epithelia co-cultured on human (Hs58 and CCD1112Sk) and mouse (3T3) fibroblasts.

Article Snippet: Immunofluorescent staining were performed with our previous methods , , , using primary antibodies against p63 , integrin β1, EGFR, K14, K3, connexin 43 (Calbiochem, Labvision, ICN Pharmaceuticals, Santa Cruz Biotechnology, Chemicon International, or Invitrogen, respectively), with Alexa Fluor 488 conjugated secondary antibodies (Invitrogen), and counter-staining with Hoechst 33342 (Sigma).

Techniques:

Journal: Drug Design, Development and Therapy

Article Title: A Potent EGFR Inhibitor, N-Phenyl Pyrazoline Derivative Suppresses Aggressiveness and Cancer Stem Cell-Like Phenotype of Cervical Cancer Cells

doi: 10.2147/DDDT.S350913

Figure Lengend Snippet: The N - phenyl pyrazoline 5 acts as a potent EGFR inhibitor. ( A ) The active site of EGFR is identified based on the erlotinib binding site to EGFR. The figure shows the overlapping of the binding conformation of erlotinib (blue) and N - phenyl pyrazoline 5 (red) in EGFR protein. ( B and C ) Both erlotinib and N - phenyl pyrazoline 5 bind to EGFR through several amino acid residues. ( D ) The Western blotting result shows that N-phenyl pyrazoline 5 reduced the EGFR expression level in a dose-dependent manner. The downstream protein of the EGFR, the total ERK1/2 expression was not affected by the EGFR inhibition.

Article Snippet: The membranes were washed out using PBS-Tween-20 (PBST), then incubated with primary antibodies specific against EGFR (A11352, Abclonal), ERK1/2 (A16686, Abclonal), CD133 (A0219, Abclonal), and β-actin (AC026, Abclonal) for overnight at 4°C.

Techniques: Binding Assay, Western Blot, Expressing, Inhibition

Journal: Drug Design, Development and Therapy

Article Title: A Potent EGFR Inhibitor, N-Phenyl Pyrazoline Derivative Suppresses Aggressiveness and Cancer Stem Cell-Like Phenotype of Cervical Cancer Cells

doi: 10.2147/DDDT.S350913

Figure Lengend Snippet: The N-phenyl pyrazoline 5 suppresses the cervical cancer stem cell-like phenotype via EGFR inhibition. ( A ) The Pearson correlation test was performed using 294 cervical cancer patients. The EGFR expression is positively associated with cancer stem cell marker expressions, including CD44, MYC, ALDH1A3, and KLF4 with r-value 0.51, 0.39, 0.34, 0.19, respectively. ( B ) The hanging drop tumorsphere assay was performed with 50 cells and 100 cells density for each drop for 7 days incubation. The N-phenyl pyrazoline 5 treatments reduce the tumorsphere size in a dose-dependent manner. ( C ) The quantification of tumorsphere size from the previous figure. ( D ) The Western blotting assay was performed to assess the expression of the cancer stem cell marker, CD133. The result shows that CD133 was decreased after the N-phenyl pyrazoline 5 treatment. *p-value < 0.05, **p < 0.01, ***p < 0.001. Scale bar: 100µm.

Article Snippet: The membranes were washed out using PBS-Tween-20 (PBST), then incubated with primary antibodies specific against EGFR (A11352, Abclonal), ERK1/2 (A16686, Abclonal), CD133 (A0219, Abclonal), and β-actin (AC026, Abclonal) for overnight at 4°C.

Techniques: Inhibition, Expressing, Marker, Incubation, Western Blot