Journal: Cells

Article Title: Molecular Analysis of the Superior Efficacy of a Dual Epidermal Growth Factor Receptor (EGFR)-DNA-Targeting Combi-Molecule in Comparison with Its Putative Prodrugs 6-Mono-Alkylamino- and 6,6-Dialkylaminoquinazoline in a Human Osteosarcoma Xenograft Model

doi: 10.3390/cells12060914

Figure Lengend Snippet: Immunohistochemistry and Western blot analysis of the effects of ZR2002, JS84 and JS61 on tumor tissues. ZR2002 decreases the phosphorylation of EGFR (P-EGFR). ( A ) Representative sections of Saos-2 tumors stained for P-EGFR. ( B ) Western blots for P-EGFR (Tyr 1068) and EGFR, where β-actin was used as internal control. Each band corresponds to different tumor lysates. ( C ) Quantification of Western blot: ratio of band intensity between P-EGFR and EGFR total protein. Data are expressed as means ± SEM. * p < 0.05 between the two groups.

Article Snippet: The membranes were blocked with 5% BSA in PBS 1x with 0.1% Tween-20 and immunoblotted overnight with the following antibodies—P-EGFR (Tyr1068) (D7A5) (1:1000; #3777; Cell Signaling, Danvers, MA, USA), EGFR (1:1000; #2646; Cell Signaling, Danvers, MA, USA) and phospho-histone H2A.X (Ser139) (1:1000; #2577; Cell Signaling, Danvers, MA, USA)—for tumor-tissue-derived blots and immunoblotted with the EGFR antibody for tumor-cell-derived blots.

Techniques: Immunohistochemistry, Western Blot, Staining

Journal: Redox Biology

Article Title: NADPH oxidase 1: A target in the capacity of dimeric ECG and EGCG procyanidins to inhibit colorectal cancer cell invasion

doi: 10.1016/j.redox.2023.102827

Figure Lengend Snippet: ECG and EGCG dimers inhibited EGF-mediated activation of the EGFR and downstream of NF-κB, Akt, and ERK1/2 pathways. Caco-2 cells were pre-incubated with or without ECG and EGCG dimers for 30 min and then with or without EGF (10 ng/ml) for subsequent 10 min. Phosphorylation levels of B- EGFR (Tyr1068), C– IKK (Ser176/180), D- p65 (Ser536), G- Akt (Ser473), and H- ERK1/2 ((Thr202/Tyr204) were evaluated by Western blot. A, F- Representative Western blot images. Bands were quantified and values for phosphorylated proteins were referred to the respective total protein content. E− NF-κB activation was also evaluated by EMSA measuring NF-κB-DNA binding in nuclear fractions. Results are shown as means ± SEM of 3–5 independent experiments. Values having different superscripts are significantly different (p < 0.05, One-way ANOVA-test).

Article Snippet: Primary antibodies for p(Tyr1068)-EGFR (#3777), EGFR (#4267), p(Ser176/180)-IKKα/β (2697), IKKα (#2682), p(Ser536)-p65 (#3033), p65 (#8242), p(Ser473)-Akt (#4060), Akt (#4691), p(Thr202, Tyr204)-ERK (#4370), ERK (#9102) and β-actin (#12620); secondary antibodies anti-rabbit HRP-conjugated (#7074), anti-rabbit biotinylated (#14708), streptavidin (#3999) and the biotinylated ladder (#7727) were from Cell Signaling Technology, Inc. (Danvers, MA).

Techniques: Activation Assay, Incubation, Western Blot, Binding Assay

Journal: Redox Biology

Article Title: NADPH oxidase 1: A target in the capacity of dimeric ECG and EGCG procyanidins to inhibit colorectal cancer cell invasion

doi: 10.1016/j.redox.2023.102827

Figure Lengend Snippet: NADPH oxidase inhibitors and ECG and EGCG dimers inhibited EGF-mediated oxidant production and the EGFR signaling pathway in Caco-2 cells. Caco-2 cells were pre-incubated with or without 10 μM ECG, 30 μM EGCG, 1 μM apocynin, 1 μM Vas or 1 μM DPI dimers for 30 min and then with or without EGF (10 ng/ml) for subsequent 10 min or 6 h. A-C- Oxidant levels were measured using the probes A- DHE, B- DCF and C- Amplex Red as described in methods. D- NOX1 mRNA levels were measured by qPCR and referred to actin mRNA levels as housekeeping gene. E-J Phosphorylation levels of F- EGFR (Tyr1068), G- IKK (Ser176/180), H-p65 (Ser536), J- Akt (Ser473), and ERK1/2 (Thr202/Tyr204) were evaluated by Western blot. E, I- Representative Western blot images. Bands were quantified and values for phosphorylated proteins were referred to the respective total protein content. Results are shown as means ± SEM of 3–5 independent experiments. Values having different superscripts are significantly different (p < 0.05, One-way ANOVA-test). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Primary antibodies for p(Tyr1068)-EGFR (#3777), EGFR (#4267), p(Ser176/180)-IKKα/β (2697), IKKα (#2682), p(Ser536)-p65 (#3033), p65 (#8242), p(Ser473)-Akt (#4060), Akt (#4691), p(Thr202, Tyr204)-ERK (#4370), ERK (#9102) and β-actin (#12620); secondary antibodies anti-rabbit HRP-conjugated (#7074), anti-rabbit biotinylated (#14708), streptavidin (#3999) and the biotinylated ladder (#7727) were from Cell Signaling Technology, Inc. (Danvers, MA).

Techniques: Incubation, Western Blot

Journal: Redox Biology

Article Title: NADPH oxidase 1: A target in the capacity of dimeric ECG and EGCG procyanidins to inhibit colorectal cancer cell invasion

doi: 10.1016/j.redox.2023.102827

Figure Lengend Snippet: Silencing of NOX1 inhibited EGF-induced cell invasion, MMP-2 and MMP-9 overexpression, signaling pathways downstream the EGFR in Caco-2 cells, and cell invasion. Caco-2 cells were transfected with or without scramble (si-scramble) or NOX1 (si-NOX1) silence RNAs, for 48 h, and subsequently incubated with or without EGF (10 ng/ml) for A-D, 6 h, E− 10 min and F-24 h. A- NOX1 mRNA levels and B- NOX1 protein levels were evaluated by PCR and Western blot, respectively. C- MMP-2 and D- MMP-9 mRNA levels were measured by qPCR and referred to actin mRNA levels as housekeeping gene. E− Phosphorylation levels of EGFR (Tyr1068), IKK (Ser176/180), p65 (Ser536), Akt (Ser473) and ERK1/2 (Thr202/Tyr204) were evaluated by Western blot. Bands were quantified and values for phosphorylated proteins were referred to the respective total protein content. (F) Caco-2 cells were transfected with si-scramble or si-NOX1 RNAs, and 48 h later replated into Matrigel chambers. Cells were then stimulated with EGF (10 ng/ml) for 24 h, and cell invasiveness was evaluated (20X magnification). Results are shown as means ± SEM of 3–5 independent experiments. Values having different superscripts are significantly different (p < 0.05, One-way ANOVA-test).

Article Snippet: Primary antibodies for p(Tyr1068)-EGFR (#3777), EGFR (#4267), p(Ser176/180)-IKKα/β (2697), IKKα (#2682), p(Ser536)-p65 (#3033), p65 (#8242), p(Ser473)-Akt (#4060), Akt (#4691), p(Thr202, Tyr204)-ERK (#4370), ERK (#9102) and β-actin (#12620); secondary antibodies anti-rabbit HRP-conjugated (#7074), anti-rabbit biotinylated (#14708), streptavidin (#3999) and the biotinylated ladder (#7727) were from Cell Signaling Technology, Inc. (Danvers, MA).

Techniques: Over Expression, Transfection, Incubation, Western Blot

Journal: Nature

Article Title: Injury prevents Ras mutant cell expansion in mosaic skin

doi: 10.1038/s41586-023-06198-y

Figure Lengend Snippet: a) Schematic representation of the EGFR signaling pathway. Hras G12V is constitutively active and therefore less dependent on upstream activation of EGFR. b) Western blot analysis of p-EGFR(phospho-Tyr1068) normalized on total-EGFR (n = 6 mice) and of p-ERK1/2(phospho-Thr202/Tyr204) and p-AKT(phospho-Ser473) normalized on total-ERK1/2 and total-AKT (n = 3 mice). Unpaired, two-tailed t-test . c) Mitotic figure quantification in tdTomato+ and tdTomato− areas in Hras G12V/+ -mosaic without or with EGFR-Dominant Negative (DN) expression (EGFR-WT or EGFR-DN). n = 3 mice. Unpaired or Paired two-tailed t-test for comparison between different or the same groups of mice. d) Revisit images of the same area of the basal stem cell layer. ( left ) The increase of tdTomato+ area. n = 3 mice. Unpaired, two-tailed t-test . e) Initial percent tdTomato+ area in the first revisit of injured-Hras G12V/+ -mosaic treated with vehicle (n = 3 mice) or Gefitinib (n = 4 mice). f) Epidermal preparation immunofluorescence for phospho-Histone3 in wild-type ( left ) and constitutive-p21 null ( right ) at postnatal-day-25. g) Phospho-Histone3+ cell quantification in wild-type, constitutive-p21 null with or without LSL-tdTomato at postnatal-day-25 (n = 3 mice). Unpaired, two-tailed t-test . h) Whole mount immunofluorescence for phospho-Histone3 in wild-type ( left ) and constitutive-p21 null ( right ) at postnatal-day-25. Phospho-Histone3+ dermal cell quantification (n = 4 mice). Unpaired, two-tailed t-test . i) Initial percent tdTomato+ area in the first revisit of uninjured wild-type-mosaic (n = 3 mice), Hras G12V/+ -mosaic (n = 3 mice) and constitutive-p21 null -Hras G12V/+ -mosaic (n = 4 mice). j) Keratin10+ cell quantification in tdTomato+ and tdTomato− areas in Hras G12V/+ -mosaic and constitutive-p21 null -Hras G12V/+ -mosaic in uninjured(U) ears (n = 3 mice). Unpaired or Paired two-tailed t-test for comparison between different or the same groups of mice. Exact p-values reported on the figure. ns indicates not statistically significant. At least three independent areas of approximately 300 μm 2 were analysed for each mouse . Data are represented as means and standard deviations. Scale bar, 20 μm.

Article Snippet: The following rabbit primary antibodies were used at the given concentrations; p-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (1:500, Cell Signaling 9101), p44/42 MAPK (ERK1/2) (1:500, Cell Signaling 4695), p-EGFR (Tyr1068) (1:100, Cell Signaling 2234), EGFR (1:100 Cell Signaling 4267; Extended Data Fig. ), EGFR (1:100, Cell Signaling 2232; Fig. ), p-AKT (Ser473) (1:200, Cell Signaling 4060), AKT (1:200, Cell Signaling 9262) and GAPDH (14C10) (1:500, Cell Signaling 2118).

Techniques: Activation Assay, Western Blot, Two Tailed Test, Dominant Negative Mutation, Expressing, Immunofluorescence

Journal: iScience

Article Title: IGFBP3 induced by the TGF-β/EGFRvIII transactivation contributes to the malignant phenotype of glioblastoma

doi: 10.1016/j.isci.2023.106639

Figure Lengend Snippet:

Article Snippet: Rabbit mAb anti-p-EGFR (Tyr1068) (D7A5) , Cell Signaling Technology , Cat#3777; RRID: AB_2096270.

Techniques: Luciferase, Microarray, Recombinant, Lysis, Transfection, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Reporter Gene Assay, BIA-KA, Cell Cycle Assay, Sequencing, shRNA, Real-time Polymerase Chain Reaction, Plasmid Preparation, Software