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: Oncotarget

Article Title: CHIP is a novel tumor suppressor in pancreatic cancer and inhibits tumor growth through targeting EGFR

doi:

Figure Lengend Snippet: (A) The levels of proteins that were degraded by CHIP in other types of cells were determined by immunoblotting. (B) CHIP interacts with EGFR in an endogenous or exogenous way. (i)BxPC-3 cells were pretreated with MG132 (5 μM) for 6 h, and endogenous CHIP-EGFR interaction was examined by immunoprecipitation with CHIP or EGFR antibody. (ii)BxPC-3 cells were transfected with His-EGFR and Flag-CHIP, MG132 were used after 48 h of transfection, and two exogenous protein interactions were determined by His or Flag antibody. (C) CHIP promotes EGFR degradation in Panc-1 and BxPC-3 cells. The levels of EGFR were determined after cells were infected with scrambled controls, CHIP amiRNA or CHIP overexpression(CHIP OE ) lentiviruses. (D) CHIP enhances EGFR degradation in concentration and ubiquitination/proteasome dependent ways. BxPC-3 cells were co-transfected with HA-ubiquitin, His-EGFR and Flag-CHIP plasmids (0, 1 μg, 2 μg per well in a six-well plate). The levels of His-EGFR were determined by immunoblotting, using antibody against His-tag. Wells in another six-well plate were treated with geldanamycin (GA,1 μM) or MG132 (5 μM) for 6 h after 48 h co-transfection. The levels of His-EGFR and HA-ubiquitin were detected with anti-His or anti-HA antibody. (E) CHIP enhances EGFR degradation in a time-dependent manner. BxPC-3 cells were transfected with control,CHIP OE or CHIP amiRNA for 0, 4, 8, 12, or 24 h. The levels of EGFR were determined by immunoblotting.

Article Snippet: Antibodies and their sources were as follows: anti-CHIP antibody (Santa Cruz,California,USA); anti-EGFR antibody and anti-phosphorylated EGFR (Tyr845, Tyr1068, Tyr1173) antibody (Cell Signaling,Massachusettes,USA); anti-AKT antibody and anti-phosphorylated AKT (Ser473) antibody (Cell Signaling); anti-mTOR antibody and anti-phosphorylated mTOR (Ser2448) antibody (Cell Signaling); anti BAD antibody and anti-phosphorylated BAD (Ser136) antibody (Cell Signaling); anti-p21 antibody (Cell Signaling); anti-Src antibody and anti-phosphorylated Src (Tyr416) antibody (Cell Signaling); anti-FAK antibody and anti-phosphorylated FAK (Tyr 925) antibody (Cell Signaling); anti-paxillin antibody and anti-phosphorylated paxillin (Tyr118) antibody (Cell Signaling); anti-Erk1/2 antibody and anti-phosphorylated Erk1/2 (Thr202/Tyr204) antibody (Cell Signaling); anti-His antibody (Santa Cruz); anti-Flag antibody (Santa Cruz); anti ki67 antibody (Abcam,Cambridge,UK); and anti-Cleaved Caspase-3 antibody (Cell Signaling).

Techniques: Western Blot, Immunoprecipitation, Transfection, Infection, Over Expression, Concentration Assay, Cotransfection

Journal: Oncotarget

Article Title: CHIP is a novel tumor suppressor in pancreatic cancer and inhibits tumor growth through targeting EGFR

doi:

Figure Lengend Snippet: (A) (i)Two main functional domains of CHIP are illustrated schematically. (ii) The U-box domain of CHIP is required for degradation of EGFR. BxPC-3 cells were co-transfected with HA-ubiquitin, His-EGFR and Flag tagged CHIP-full length (CHIP FL ) or its Flag tagged domains (CHIP ΔU-box for CHIP protein without a U-box domain, CHIP ΔTPR for CHIP protein without a TPR domain) for 48 h. Then, the cells were treated with or without MG132 (5 μM). EGFR and ubiquitin were detected by immunoblotting with anti-His or anti-HA antibody. Anti-Flag antibody was used to test the expression of CHIP FL or its domains. (iii) CHIP FL is required to interact with EGFR. BxPC-3 cells were co-transfected with His-EGFR and Flag-CHIP FL , Flag-CHIP ΔU-box or Flag-CHIP ΔTPR for 48 h; then, the cells were treated with MG132 (5 μM). CHIP or its domains were combined by anti-Flag antibody (IP) and immunoprecipitated by A/G agarose beads. Immunoblotting (IB) using anti-His antibody was performed to determine the exogenous expression of EGFR. (B) The downstream pathways of EGFR are regulated by CHIP in Panc-1 and BxPC-3 cells. The lysate of stable CHIP knockdown cells or CHIP OE cells were used to determine the expression of AKT/mTOR, Src/FAK/paxillin,MAPK pathways by different antibodies. In the figure, p- represents phosphorylated, and t- represents total. (C) CHIP down-regulates phosphorylation of Tyr845 and Tyr 1068 of EGFR in Panc-1 and BxPC-3 cells. (D) CHIP is co-localized with EGFR in Bxpc-3 cells and attenuates the expression of EGFR. BxPC-3 cells were transfected with vector or Flag-CHIP plasmid combined with His-EGFR plasmid. After 48 h, the cells were treated with or without EGF (50 ng/mL for 30 min) and then were stained with anti-His or anti-Flag antibody. The white arrows indicate that EGFR was present or absent due to CHIP under- or over-expression in the cytoplasm or nucleus.

Article Snippet: Antibodies and their sources were as follows: anti-CHIP antibody (Santa Cruz,California,USA); anti-EGFR antibody and anti-phosphorylated EGFR (Tyr845, Tyr1068, Tyr1173) antibody (Cell Signaling,Massachusettes,USA); anti-AKT antibody and anti-phosphorylated AKT (Ser473) antibody (Cell Signaling); anti-mTOR antibody and anti-phosphorylated mTOR (Ser2448) antibody (Cell Signaling); anti BAD antibody and anti-phosphorylated BAD (Ser136) antibody (Cell Signaling); anti-p21 antibody (Cell Signaling); anti-Src antibody and anti-phosphorylated Src (Tyr416) antibody (Cell Signaling); anti-FAK antibody and anti-phosphorylated FAK (Tyr 925) antibody (Cell Signaling); anti-paxillin antibody and anti-phosphorylated paxillin (Tyr118) antibody (Cell Signaling); anti-Erk1/2 antibody and anti-phosphorylated Erk1/2 (Thr202/Tyr204) antibody (Cell Signaling); anti-His antibody (Santa Cruz); anti-Flag antibody (Santa Cruz); anti ki67 antibody (Abcam,Cambridge,UK); and anti-Cleaved Caspase-3 antibody (Cell Signaling).

Techniques: Functional Assay, Transfection, Western Blot, Expressing, Immunoprecipitation, Plasmid Preparation, Staining, Over Expression

Journal: Oncotarget

Article Title: CHIP is a novel tumor suppressor in pancreatic cancer and inhibits tumor growth through targeting EGFR

doi:

Figure Lengend Snippet: (A) CHIP suppresses cell growth rate. CHIP amiRNA, CHIP OE and their corresponding control Panc-1 or BxPC-3 cells were grown in 96-well plates for 1, 2, 3, 4, 5 and 6 days. Cell survival was detected by CCK-8 analysis (mean±standard deviation; **P<.01). (B) CHIP suppresses anchorage-independent growth in Panc-1 and BxPC-3 cells. Stable CHIP knockdown or CHIP OE cells were plated in a 6-well plate that contained soft agar. After incubation for 21 days, colonies were photographed and counted under the microscope (mean±standard deviation; *P<.05, **P<.01). (C)The stable CHIP knockdown or CHIP OE cells and their control BxPC-3 cells were subcutaneously injected into nude mice. Thirty-seven days after the injections, the mice were sacrificed, and tumor tissues were collected. The left panel shows tumor growth curves in nude mice; the middle and right panel shows the size and weight of the tumors after 37 days (mean±standard deviation; *P<.05, **P<.01). (D) CHIP OE decreases, but knockdown CHIP enhances the expression of EGFR and Ki67. Sections of tumors from injected nude mice were stained with CHIP, EGFR and Ki67 antibodies by immunohistochemistry (magnification ×100). The right panel shows the percentage of strongly Ki67 stained tumor cells (mean±standard deviation; *P<.05).

Article Snippet: Antibodies and their sources were as follows: anti-CHIP antibody (Santa Cruz,California,USA); anti-EGFR antibody and anti-phosphorylated EGFR (Tyr845, Tyr1068, Tyr1173) antibody (Cell Signaling,Massachusettes,USA); anti-AKT antibody and anti-phosphorylated AKT (Ser473) antibody (Cell Signaling); anti-mTOR antibody and anti-phosphorylated mTOR (Ser2448) antibody (Cell Signaling); anti BAD antibody and anti-phosphorylated BAD (Ser136) antibody (Cell Signaling); anti-p21 antibody (Cell Signaling); anti-Src antibody and anti-phosphorylated Src (Tyr416) antibody (Cell Signaling); anti-FAK antibody and anti-phosphorylated FAK (Tyr 925) antibody (Cell Signaling); anti-paxillin antibody and anti-phosphorylated paxillin (Tyr118) antibody (Cell Signaling); anti-Erk1/2 antibody and anti-phosphorylated Erk1/2 (Thr202/Tyr204) antibody (Cell Signaling); anti-His antibody (Santa Cruz); anti-Flag antibody (Santa Cruz); anti ki67 antibody (Abcam,Cambridge,UK); and anti-Cleaved Caspase-3 antibody (Cell Signaling).

Techniques: CCK-8 Assay, Standard Deviation, Incubation, Microscopy, Injection, Expressing, Staining, Immunohistochemistry