Journal: Journal of Carcinogenesis

Article Title: Sulindac metabolites inhibit epidermal growth factor receptor activation and expression

doi: 10.1186/1477-3163-4-16

Figure Lengend Snippet: EGF induces EGFR and ERK1/2 phosphorylation . HT29 human colon cancer cells were grown to 80% confluence in medium containing 10% FBS then serum deprived for 48 h before treatment with vehicle (water), 10, or 100 ng/ml EGF. Cells were harvested 10 min after EGF treatment and lysates prepared for (A) Immunoblotting with antibodies raised against pEGFR (pY1068), total EGFR, pERK1/2, and total ERK1/2; α-tubulin immunoblots of the same lysates served as loading controls. The graphs show the densitometry results of the pEGFR bands (B) and pERK1/2 bands (C) normalized for the loading controls. Data represent mean of triplicate samples ± SD; statistical significance is denoted by ***p < 0.001 versus vehicle. Results shown in the figure are representative of 2 separate experiments each with triplicate samples.

Article Snippet: Primary antibodies raised against phosphorylated ERK1/2 (Thr202/Tyr204), total ERK1/2, and total EGFR were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); primary antibody against phosphorylated EGFR(Tyr1068), horseradish peroxidase-conjugated anti-mouse and anti-rabbit secondary antibodies, and EGF were purchased from Biosource (Camarillo, CA).

Techniques: Western Blot

Journal: Journal of Carcinogenesis

Article Title: Sulindac metabolites inhibit epidermal growth factor receptor activation and expression

doi: 10.1186/1477-3163-4-16

Figure Lengend Snippet: Dose response of sulindac sulfide inhibition of EGFR . HT29 cells were grown to 80% confluence in medium containing 10% FBS then serum deprived for 24 h before addition of drug. Cells were then treated with vehicle (0.1% DMSO), 40, 80, 120, or 160 μM sulindac sulfide, drug doses previously shown to induce apoptotic cell death in these cells. Twenty four hours after drug treatment, vehicle or 10 ng/ml EGF was added and cells were harvested 10 min later. (A) Immunoblots were performed on cell lysates with antibodies raised against pEGFR (pY1068), total EGFR, pERK1/2, total ERK1/2, and caspase-3; total ERK1/2 immunoblots served as loading controls. The graphs show the densitometry results of the pEGFR bands (B) and total EGFR bands (C) . Results shown in figure are representative of 3 separate experiments.

Article Snippet: Primary antibodies raised against phosphorylated ERK1/2 (Thr202/Tyr204), total ERK1/2, and total EGFR were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); primary antibody against phosphorylated EGFR(Tyr1068), horseradish peroxidase-conjugated anti-mouse and anti-rabbit secondary antibodies, and EGF were purchased from Biosource (Camarillo, CA).

Techniques: Inhibition, Western Blot

Journal: Journal of Carcinogenesis

Article Title: Sulindac metabolites inhibit epidermal growth factor receptor activation and expression

doi: 10.1186/1477-3163-4-16

Figure Lengend Snippet: Dose response of sulindac sulfone inhibition of EGFR . HT29 cells were grown to 80% confluence in medium containing 10% FBS then serum deprived for 24 h before addition of drug. Cells were then treated with vehicle (0.2% DMSO), 200, 400, 600, or 800 μM sulindac sulfone, drug doses previously shown to induce apoptotic cell death in these cells. Twenty four hours after drug treatment, vehicle or 10 ng/ml EGF was added and cells were harvested 10 min later. (A) Immunoblots were performed on cell lysates with antibodies raised against pEGFR (pY1068), total EGFR, pERK1/2, total ERK1/2, and caspase-3; total ERK1/2 immunoblots served as loading controls. The graphs show the densitometry results of the pEGFR bands (B) and total EGFR bands (C) . Results shown in figure are representative of 3 separate experiments.

Article Snippet: Primary antibodies raised against phosphorylated ERK1/2 (Thr202/Tyr204), total ERK1/2, and total EGFR were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); primary antibody against phosphorylated EGFR(Tyr1068), horseradish peroxidase-conjugated anti-mouse and anti-rabbit secondary antibodies, and EGF were purchased from Biosource (Camarillo, CA).

Techniques: Inhibition, Western Blot

Journal: Journal of Carcinogenesis

Article Title: Sulindac metabolites inhibit epidermal growth factor receptor activation and expression

doi: 10.1186/1477-3163-4-16

Figure Lengend Snippet: Dose response and time course of sulindac sulfide inhibition of EGFR . HT29 cells were grown to confluence in medium containing 10% FBS and treated with vehicle (0.1% DMSO), 160, or 180 μM sulindac sulfide for 1 h, 12 h, and 24 h. Cells were then harvested and immunoblots were performed on cell lysates with antibodies raised against pEGFR (pY1068), total EGFR, pERK1/2, total ERK1/2, and cleaved caspase-3; α-tubulin immunoblots of the same lysates served as loading controls. (A) 1 h, 12 h, and 24 h immunoblot results. The graphs show the densitometry results of the pEGFR bands (B) and total EGFR bands (C) . Data represent mean of triplicate samples ± SD; statistical significance is denoted by **p < 0.01 and ***p < 0.001 versus respective time point vehicle. Results shown in figure are representative of 2 separate experiments each with triplicate samples.

Article Snippet: Primary antibodies raised against phosphorylated ERK1/2 (Thr202/Tyr204), total ERK1/2, and total EGFR were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); primary antibody against phosphorylated EGFR(Tyr1068), horseradish peroxidase-conjugated anti-mouse and anti-rabbit secondary antibodies, and EGF were purchased from Biosource (Camarillo, CA).

Techniques: Inhibition, Western Blot

Journal: Journal of Carcinogenesis

Article Title: Sulindac metabolites inhibit epidermal growth factor receptor activation and expression

doi: 10.1186/1477-3163-4-16

Figure Lengend Snippet: Dose response and time course of sulindac sulfone inhibition of EGFR . HT29 cells were grown to confluence in medium containing 10% FBS followed by treatment with vehicle (0.2% DMSO), 400, or 600 μM sulindac sulfone for 1 h, 12 h, and 24 h. Cells were then harvested and immunoblots were performed on cell lysates with antibodies raised against pEGFR (pY1068), total EGFR, pERK1/2, total ERK1/2, and cleaved caspase-3; α-tubulin immunoblots of the same lysates served as loading controls. (A) 1 h, 12 h, and 24 h Western blot results. The graphs show the densitometry results of the pEGFR bands (B) and total EGFR bands (C) . Data represent mean of triplicate samples ± SD; statistical significance is denoted by **p < 0.01 and ***p < 0.001 versus respective time point vehicle. Results shown in figure are representative of 2 separate experiments each with triplicate samples.

Article Snippet: Primary antibodies raised against phosphorylated ERK1/2 (Thr202/Tyr204), total ERK1/2, and total EGFR were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); primary antibody against phosphorylated EGFR(Tyr1068), horseradish peroxidase-conjugated anti-mouse and anti-rabbit secondary antibodies, and EGF were purchased from Biosource (Camarillo, CA).

Techniques: Inhibition, Western Blot

Journal: Journal of Carcinogenesis

Article Title: Sulindac metabolites inhibit epidermal growth factor receptor activation and expression

doi: 10.1186/1477-3163-4-16

Figure Lengend Snippet: Effect of the caspase inhibitor, ZVAD, on apoptosis and inhibition of EGFR . HT29 colon cancer cells were grown to confluence in medium containing 10% FBS followed by pretreatment with or without 25 μM zvad for 1 h. Cells were then treated with vehicle (0.2% DMSO) or 600 μM sulfone for 48 h. Cells were harvested and immunoblots were performed on cell lysates with antibodies raised against pEGFR (pY1068), total EGFR, and cleaved caspase 3; α-tubulin immunoblots of the same lysates served as loading controls. The graphs show morphological apoptosis results (A) 48 h Western immunoblot results (B) and densitometry of the pEGFR bands (C) and total EGFR bands (D) . Data represent mean of triplicate samples ± SD; statistical significance is denoted by *p < 0.05, **p < 0.01 and ***p < 0.001. Results shown in figure are representative of 2 separate experiments each with triplicate samples.

Article Snippet: Primary antibodies raised against phosphorylated ERK1/2 (Thr202/Tyr204), total ERK1/2, and total EGFR were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); primary antibody against phosphorylated EGFR(Tyr1068), horseradish peroxidase-conjugated anti-mouse and anti-rabbit secondary antibodies, and EGF were purchased from Biosource (Camarillo, CA).

Techniques: Inhibition, Western Blot

Journal: Cancer Communications

Article Title: Aldolase A promotes proliferation and G 1 /S transition via the EGFR/MAPK pathway in non-small cell lung cancer

doi: 10.1186/s40880-018-0290-3

Figure Lengend Snippet: ALDOA enhanced H520 cell proliferation and cyclin D1 expression in an epidermal growth factor receptor/mitogen-activated protein kinase (EGFR/MAPK) pathway-dependent manner. a Cyclin D1 expression was tested by western blotting after treatment with mitogen-activated kinase kinase 1/2 (MEK1/2) inhibitor U0126-EtOH or DMSO control treatment. Cyclin D1 protein was quantified by western blotting. Data are shown as mean ± SD in ( b ). β-actin was used as loading control. * P < 0.05 versus shNC. c Colony formation assays after MEK1/2 activity blockade with U0126-EtOH. The colony numbers were counted, and the rate of decrease in each group was calculated using the following formula: decrease rate = (colony number of DMSO treatment − colony number of actinomycin D treatment)/colony number of DMSO treatment. The results indicated that rate of decrease in the colony number was higher in the shNC than shAL-1 or shAL-2 group. Data are shown as mean ± SD in ( d ). * P < 0.05 versus shNC. e Cells were serum-starved for 24 h or supplemented with 50 ng/mL epidermal growth factor (EGF) for 0.5 or 1 h, respectively. Cyclin D1 and relevant proteins that are engaged in the EGFR/MAPK pathway were measured by western blotting. The results showed that cyclin D1 protein was almost the same in the shAL and shNC group cells with EGF treatment for 0.5 h. Data are shown as mean ± SD in ( f ). β-actin was used as loading control

Article Snippet: Primary antibodies for retinoblastoma protein (Rb, 1:1500), phosphorylated EGFR (p-EGFR) (Tyr1068) (1:1000), p-Raf-1 (Ser259) (1:1000) and MEK1/2 (1:10,000) were purchased from Abcam.

Techniques: Expressing, Western Blot, Activity Assay

Journal: Cancer Communications

Article Title: Aldolase A promotes proliferation and G 1 /S transition via the EGFR/MAPK pathway in non-small cell lung cancer

doi: 10.1186/s40880-018-0290-3

Figure Lengend Snippet: Mutual regulation relationship between ALDOA and epidermal growth factor receptor/mitogen-activated protein kinase (EGFR/MAPK) pathway. a Key components of the EGFR/MAPK pathway were measured by western blotting. β-actin was used as loading control. The results showed that EGFR, p-EGFR (Tyr1068), Raf-1, p-Raf-1 (Ser259) and p-MEK1/2 (Ser217/S221) were decreased and that p-ERK1/2 (Thr202/Tyr204) was upregulated in shAL cells compared with shNC cells. b Immunofluorescence assays of EGFR and p-EGFR (Tyr1068). EGFR or p-EGFR (Tyr1068) was labeled with fluorescein isothiocyanate-conjugated antibody (green), and the nucleus was labeled by 4′,6-diamidino-2-phenylindole (blue). ALDOA-knockdown H520 cells exhibited weaker staining of EGFR and p-EGFR (Tyr1068) compared with negative control cells. c ALDOA distribution after MEK1/2 inhibition. Nuclear and cytoplasmic proteins of H520 cells were separately extracted after U0126-EtOH treatment. The ALDOA and p-ERK1/2 distributions were examined by western blotting. Octamer-binding transcription factor-1 (Oct-1) or β-tublin was used as a loading control of the nucleus or the cytoplasm

Article Snippet: Primary antibodies for retinoblastoma protein (Rb, 1:1500), phosphorylated EGFR (p-EGFR) (Tyr1068) (1:1000), p-Raf-1 (Ser259) (1:1000) and MEK1/2 (1:10,000) were purchased from Abcam.

Techniques: Western Blot, Immunofluorescence, Labeling, Staining, Negative Control, Inhibition, Binding Assay