Journal: mAbs

Article Title: Structural and functional characterization of MBS301, an afucosylated bispecific anti-HER2 antibody

doi: 10.1080/19420862.2018.1486946

Figure Lengend Snippet: ADCC activity evaluation of anti-HER2 antibodies on four target cancer cells.

Article Snippet: Human breast cancer cell line BT-474 (HTB-20), MDA-MB-175VII (HTB-25), SK-BR-3(HTB-30) and HCC1419(CRL-2326) are HER2-positive cell lines, which were obtained from the ATCC, and HCC1419 is trastuzumab-resistant human breast cancer cell line.

Techniques: Activity Assay

Journal: Cancers

Article Title: NEDD9 Overexpression Causes Hyperproliferation of Luminal Cells and Cooperates with HER2 Oncogene in Tumor Initiation: A Novel Prognostic Marker in Breast Cancer

doi: 10.3390/cancers15041119

Figure Lengend Snippet: NEDD9 is overexpressed in human HER2+ breast cells and supports resistance to lapatinib. ( A ) Western blot (WB) analysis of MCF10A, BT-474, SK-BR-3, AU565, JIMT-1, and JIMT-1-Br3 (brain metastases subline of JIMT1), using anti-NEDD9, -HER2, and -GAPDH as loading controls. ( B ) WB-based quantification of NEDD9 (green) and HER2 (red) protein expression in cell lines as in ( A ). n = 3 independent experiments. Parson correlation coefficient r = 0.972, with exception of JIMT1, where expression of NEDD9 and HER2 do not correlate. ( C ) Western blot analysis of lysates of AU565, JIMT1, and BT474 cells treated with scramble (−) non-targeting control or two different shRNAs against NEDD9 (shNEDD9), using anti-NEDD9 and -GAPDH as loading controls. ( D ) Cell viability assessment using Cyquant dye with different concentrations of lapatinib, as indicated in the figure. Two-way ANOVA, Tukey’s multiple comparison +/− SEM. AU565, JIMT1, BT474, * p < 0.0001 (DMSO vs. shNEDD-1 or -2), ns—non significant. The original western blots of is .

Article Snippet: Cell lines BT-474, SK-BR-3, AU565, and MCF10A were purchased from and authenticated by American Type Culture Collection (Manassas, VA, USA), and were cultured based on the manufacturer’s recommendations.

Techniques: Western Blot, Expressing, CyQUANT Assay

Journal: International Journal of Molecular Sciences

Article Title: Expression of RBMS3 in Breast Cancer Progression

doi: 10.3390/ijms24032866

Figure Lengend Snippet: In vitro analysis of RBMS3 expression in breast cancer cell lines representing the four main molecular types of breast cancer and a control cell line (Me16C). ( a ) The statistical analysis of RBMS3 ′s expression at the mRNA level showed a significantly different expression of RBMS3 in all the examined cell lines in comparison to the control Me16C cell line. ( b , c ) Analysis at the protein level showed a significantly higher expression of RBMS3 in the MDA-MB-231 and SK-BR-3 cell lines than in the MCF-7 and BT-474 cell lines; (( a ) Bonferroni’s multiple comparison test, ( b ) Bonferroni’s multiple comparison test, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: Respective culture media were used to provide optimal conditions for cell growth: MEBM (Lonza, Basel, Switzerland) for the Me16C cell line, αMEM (Lonza) for the MCF-7 and BT-474 cell lines, McCoy’s (ATCC) for the SK-BR-3 cell line, L-15 (Lonza) for the MDA-MB-231 cell line.

Techniques: In Vitro, Expressing

Journal: International Journal of Nanomedicine

Article Title: The Combined Effect of Nanobubble-IR783-HPPH-Affibody Complex and Laser on HER2-Positive Breast Cancer

doi: 10.2147/IJN.S387409

Figure Lengend Snippet: The targeting ability of the NIHA complex. The CLSM images of BT-474 cells incubated with the NIHA complex (up) and the nanobubble-Dil-HPPH complex (down). Bar, 50 μm.

Article Snippet: The BT-474 cell line and the MDA-MB-231 cell line were purchased from the American Type Culture Collection (USA).

Techniques: Incubation

Journal: International Journal of Nanomedicine

Article Title: The Combined Effect of Nanobubble-IR783-HPPH-Affibody Complex and Laser on HER2-Positive Breast Cancer

doi: 10.2147/IJN.S387409

Figure Lengend Snippet: ( A ) Cell viability of BT-474 cells detected via CCK-8. ( B ) The apoptotic ratio of BT-474 cells detected via Annexin-V flow cytometry. ** P < 0.01 compared with the NIHA-laser group.

Article Snippet: The BT-474 cell line and the MDA-MB-231 cell line were purchased from the American Type Culture Collection (USA).

Techniques: CCK-8 Assay, Flow Cytometry

Journal: International Journal of Nanomedicine

Article Title: The Combined Effect of Nanobubble-IR783-HPPH-Affibody Complex and Laser on HER2-Positive Breast Cancer

doi: 10.2147/IJN.S387409

Figure Lengend Snippet: CLSM images of BT-474 cells in the NIHA-laser, NIHA, laser, and the control groups. The nuclei of the apoptosis cells were stained by using PI (depicted as red fluorescence). Bar, 30 μm.

Article Snippet: The BT-474 cell line and the MDA-MB-231 cell line were purchased from the American Type Culture Collection (USA).

Techniques: Staining, Fluorescence

Journal: International Journal of Nanomedicine

Article Title: Polymer–lipid hybrid anti-HER2 nanoparticles for targeted salinomycin delivery to HER2-positive breast cancer stem cells and cancer cells

doi: 10.2147/IJN.S144184

Figure Lengend Snippet: Expression of HER2 in breast cancer cells, analyzed by flow cytometry. Notes: Representative fluorescence-activated cell sorting analysis of ( A ) MDA-MB-361 and ( B ) BT-474 cells tested by ALDEFLUOR assay. In the right image (ALDH+ DEAB), cells incubated with ALDH substrate (BAAA) and the specific inhibitor of ALDH, DEAB, were used to establish the baseline fluorescence of these cells (R1) and to define the ALDEFLUOR-positive region (R2). ( C ) Percentage of HER2 positively stained cells in breast cancer cells. ( D ) HER2 mean fluorescence intensity in breast cancer cells. Data are expressed as mean ± SD (n=3). ** P <0.01. Abbreviations: ALDH, aldehyde dehydrogenase; BAAA, BODIPY-aminoacetaldehyde; DEAB, diethylaminobenzaldehyde.

Article Snippet: Human breast cancer cell lines BT-474 and MDA-MB-361 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Flow Cytometry, Fluorescence, FACS, Incubation, Staining

Journal: International Journal of Nanomedicine

Article Title: Polymer–lipid hybrid anti-HER2 nanoparticles for targeted salinomycin delivery to HER2-positive breast cancer stem cells and cancer cells

doi: 10.2147/IJN.S144184

Figure Lengend Snippet: Cell proliferation assay. Notes: ALDH+ and ALDH− breast cancer cells were seeded in 96-well plates with a density of 1×10 4 cells per well overnight. The cells were incubated with the nanoparticles or salinomycin for 72 h, and cell viability was evaluated using the CCK-8 assay. ( A ) MDA-MB-361 ALDH+, ( B ) MDA-MB-361 ALDH−, ( C ) BT-474 ALDH+, and ( D ) BT-474 ALDH−. Data are expressed as mean ± SD (n=3). Abbreviations: ALDH, aldehyde dehydrogenase; CCK-8, Cell Counting Kit-8; Sali-NP, salinomycin-loaded polymer-lipid nanoparticles; Sali-NP-HER2, salinomycin-loaded polymer–lipid hybrid anti-HER2 nanoparticles.

Article Snippet: Human breast cancer cell lines BT-474 and MDA-MB-361 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Proliferation Assay, Incubation, CCK-8 Assay, Cell Counting

Journal: International Journal of Nanomedicine

Article Title: Polymer–lipid hybrid anti-HER2 nanoparticles for targeted salinomycin delivery to HER2-positive breast cancer stem cells and cancer cells

doi: 10.2147/IJN.S144184

Figure Lengend Snippet: The IC 50 values of salinomycin and nanoparticles in breast cancer cells

Article Snippet: Human breast cancer cell lines BT-474 and MDA-MB-361 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques:

Journal: International Journal of Nanomedicine

Article Title: Polymer–lipid hybrid anti-HER2 nanoparticles for targeted salinomycin delivery to HER2-positive breast cancer stem cells and cancer cells

doi: 10.2147/IJN.S144184

Figure Lengend Snippet: Effect of treatments on the percentage of CSCs. Notes: Effect of the treatments on the proportion of CSCs in breast cancer cells, as reflected by the tumorsphere formation rate ( A and B ) and the proportion of ALDH+ cells ( E and F ). Representative images of tumorspheres formed by ( C ) MDA-MB-361 cells and ( D ) BT-474 cells are shown. The rate of tumorsphere formation is defined as the number of tumorspheres formed in 7 days in the treatment group divided by the number of tumorspheres formed in 7 days in the untreated group; the rate of tumorsphere formation in the untreated group is used as a control and defined as 100%. The two groups were compared by one-way ANOVA with the Newman–Keuls method. Data are expressed as mean ± SD (n=6). * P <0.05; ** P <0.01; *** P <0.001. Abbreviations: ANOVA, analysis of variance; ALDH, aldehyde dehydrogenase; CSCs, cancer stem cells; Sali-NP, salinomycin-loaded polymer–lipid nanoparticles; NP-HER2, polymer–lipid anti-HER2 nanoparticles; Sali-NP-HER2, salinomycin-loaded polymer–lipid hybrid anti-HER2 nanoparticles.

Article Snippet: Human breast cancer cell lines BT-474 and MDA-MB-361 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques:

Journal: International Journal of Nanomedicine

Article Title: Polymer–lipid hybrid anti-HER2 nanoparticles for targeted salinomycin delivery to HER2-positive breast cancer stem cells and cancer cells

doi: 10.2147/IJN.S144184

Figure Lengend Snippet: In vivo antitumor assay in mice bearing subcutaneous BT-474 tumors. Notes: BT-474 tumors reached ~50 mm 3 in size by day 10. From day 10, mice were injected with nanoparticles (7.5 mg salinomycin/kg, i.v.) through the tail vein, and salinomycin (7.5 mg salinomycin/kg) dissolved in ethanol was administered by intraperitoneal injection. Therapy was given nine times on alternate days (indicated by arrows), and tumor volume was calculated. ( A ) Tumor growth curve. ( B ) Excised tumors. ( C ) The excised tumors were weighed at the end point. On day 28, the effect of the drug treatments on the CSC proportion in BT-474 tumors in vivo was evaluated by ( D ) the rate of tumorsphere formation and ( E ) the proportion of ALDH+ cells from the excised tumors. ( F ) Representative images of tumorspheres from ( D ) are shown. The two groups were compared by one-way ANOVA with the Newman–Keuls method. Data are expressed as mean ± SD (n=8). * P <0.05; ** P <0.01; *** P <0.001. Abbreviations: ANOVA, analysis of variance; ALDH, aldehyde dehydrogenase; CSC, cancer stem cell; i.v., intravenous; Sali-NP, salinomycin-loaded polymer–lipid nanoparticles; NP-HER2, polymer–lipid anti-HER2 nanoparticles; Sali-NP-HER2, salinomycin-loaded polymer–lipid hybrid anti-HER2 nanoparticles.

Article Snippet: Human breast cancer cell lines BT-474 and MDA-MB-361 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: In Vivo, Injection

Journal: Breast Cancer Research : BCR

Article Title: NVP-AUY922: a small molecule HSP90 inhibitor with potent antitumor activity in preclinical breast cancer models

doi: 10.1186/bcr1996

Figure Lengend Snippet: Protein expression analysis in a panel of human breast cancer cell lines and effect of NVP-AUY922 on the HSP90-p23 complex in BT-474 cells. (a) The expression of HSP90 and specific proteins affected by HSP90 inhibition (Her3 [EGFR3], Her2 [ErbB2, EGFR2], phospho Her2 [pHer2], EGFR, AKT [PKB], phospho-AKT [pAkt], estrogen receptor-alpha [ERa], PI3K [p110α], PDK1, HSP70, Hsc70, Rb, pMEK1/2, pERK, Bax, Bcl-2, Bad, and Bcl-XL) was analyzed in seven human breast cancer cell lines (BT20, BT-474, MDA-MB-157 [MB-157], MDA-MB-231 [MB-231], MDA-MB-468 [MB-468], SKBr3, and MCF-7) by Western blot analysis. (b) The ERBB2-overexpressing and estrogen receptor-positive cell line BT-474 was chosen for studies of the kinetics and concentration-dependent dissociation of HSP90-p23 complexes and client proteins in the presence of NVP-AUY922 or tanespimycin (17-AAG). The amount of p23 associated with HSP90 was determined by immunoprecipitating p23 followed by immunoblotting for HSP90. The levels of ERBB2, AKT, phosphorylated AKT and β-tubulin were determined by immunoblotting. DMSO, dimethyl sulfoxide; HSP90, heat shock protein 90.

Article Snippet: The ER-positive ERBB2-overexpressing cell line BT-474, which initially was derived from a human breast ductal carcinoma established from a solid invasive ductal carcinoma of the breast of a 60-year-old woman, was purchased from the ATCC (HTB-20).

Techniques: Expressing, Inhibition, Western Blot, Concentration Assay

Journal: Breast Cancer Research : BCR

Article Title: NVP-AUY922: a small molecule HSP90 inhibitor with potent antitumor activity in preclinical breast cancer models

doi: 10.1186/bcr1996

Figure Lengend Snippet: Pharmacokinetic/pharmacodynamic analysis of NVP-AUY922 in BT-474 tumor-bearing nude mice. A pharmacokinetic profile of NVP-AUY922 in BT-474 tumor xenografts, plasma, and organs (liver, heart, lung, and muscle) after administration of a single dose is shown. Female athymic mice bearing subcutaneous xenotransplants of the human ductal breast carcinoma BT-474 of approximately 250 mm 3 received a single dose of 30 mg/kg of NVP-AUY922 (intravenous) at 0 hours. (a) NVP-AUY922 concentrations were determined by HPLC/MS-MS analysis using an internal standard method. The limits of quantification for plasma and tissues were set to 4.0 ng/mL and 10 ng/g, respectively. Bars represent standard error of the mean (n = 4). (b) Pharmacodynamic analysis in BT-474 xenografts following a single NVP-AUY922 dose. The amount of p23 associated with HSP90 was determined by immunoprecipitating HSP90 followed by immunoblotting for p23. The levels of AKT, phosphorylated AKT and β-tubulin were determined by immunoblotting. HPLC/MS-MS, high-pressure liquid chromatography/tandem mass spectrometry; HSP90, heat shock protein 90; IP, immunoprecipitation.

Article Snippet: The ER-positive ERBB2-overexpressing cell line BT-474, which initially was derived from a human breast ductal carcinoma established from a solid invasive ductal carcinoma of the breast of a 60-year-old woman, was purchased from the ATCC (HTB-20).

Techniques: Tandem Mass Spectroscopy, Western Blot, High Performance Liquid Chromatography, Mass Spectrometry, Immunoprecipitation

Journal: Breast Cancer Research : BCR

Article Title: NVP-AUY922: a small molecule HSP90 inhibitor with potent antitumor activity in preclinical breast cancer models

doi: 10.1186/bcr1996

Figure Lengend Snippet: Analysis of the kinetics of HSP70 induction and ERBB2 degradation following a singe dose of NVP-AUY922. BT-474 tumor-bearing animals were administered 50 mg/kg NVP-AUY922 at 0 hours. (a) The protein levels of HSP70 were determined by immunohistochemistry during the following week. (b) The quantification of the protein levels of HSP70. (c) ERBB2 protein levels were determined by immunohistochemistry and Western blot analysis. (d) The lowest single dose of NVP-AUY922 which mediates HSP90-p23 dissociation and reduced AKT phosphorylation was determined by immunoprecipitation and Western blot analysis. HSP70, heat shock protein 70; HSP90, heat shock protein 90.

Article Snippet: The ER-positive ERBB2-overexpressing cell line BT-474, which initially was derived from a human breast ductal carcinoma established from a solid invasive ductal carcinoma of the breast of a 60-year-old woman, was purchased from the ATCC (HTB-20).

Techniques: Immunohistochemistry, Western Blot, Immunoprecipitation

Journal: Breast Cancer Research : BCR

Article Title: NVP-AUY922: a small molecule HSP90 inhibitor with potent antitumor activity in preclinical breast cancer models

doi: 10.1186/bcr1996

Figure Lengend Snippet: Antitumor effect and tolerability of NVP-AUY922 against BT-474 tumor-bearing nude mice when administered once per week

Article Snippet: The ER-positive ERBB2-overexpressing cell line BT-474, which initially was derived from a human breast ductal carcinoma established from a solid invasive ductal carcinoma of the breast of a 60-year-old woman, was purchased from the ATCC (HTB-20).

Techniques:

Journal: Breast Cancer Research : BCR

Article Title: NVP-AUY922: a small molecule HSP90 inhibitor with potent antitumor activity in preclinical breast cancer models

doi: 10.1186/bcr1996

Figure Lengend Snippet: Antitumor effect and tolerability of NVP-AUY922 in a BT-474 human breast cancer xenograft model. BT-474 cells were inoculated subcutaneously in female nude mice carrying an estrogen-release pellet. When the tumors reached 100 to 200 mm 3 , drug treatment was initiated. Each group consisted of eight animals. NVP-AUY922 was administered (a) as a single injection, (b) three times per week (3qw), or (c) once per week (qw) at the indicated dose levels. Tumor volumes and body weights were measured three times per week. Each point represents the mean ± standard error of the mean (SEM). Arrows in right panels indicate NVP-AUY922 treatment days. Asterisks indicate statistical significance compared with vehicle-treated controls ( P < 0.05, one-way analysis of variance post hoc Dunnett). i.v., intravenous.

Article Snippet: The ER-positive ERBB2-overexpressing cell line BT-474, which initially was derived from a human breast ductal carcinoma established from a solid invasive ductal carcinoma of the breast of a 60-year-old woman, was purchased from the ATCC (HTB-20).

Techniques: Injection

Journal: Molecular Cancer

Article Title: Phosphorylation of Ser 78 of Hsp27 correlated with HER-2/ neu status and lymph node positivity in breast cancer

doi: 10.1186/1476-4598-6-52

Figure Lengend Snippet: Effect of heregulin α1 (HRG α1) and p38 MAPK inhibitor (SB 203580) on Hsp27 phosphorylation . Cultures of cells from the BT474 breast cancer cell line were treated with HRG α1 for 10 and 30 min (A) or SB 203580 for 10 hours (B) and total cell lysates were extracted using M-PER reagent (Pierce). Equal amounts of proteins (20 μg) were separated by SDS-PAGE and transferred onto PVDF membrane. The membranes were blocked for 1 hour, followed by being probed with the respective primary antibodies (anti-Hsp27, anti-pSer 15 , anti-pSer 78 and anti-pSer 82 ), and HRP-conjugated secondary antibody. The signals were captured and their intensities were detected as described in Figure 2. The phosphorylation levels of pSer 15 , pSer 78 and pSer 82 were expressed as the ratios of intensity probed with phosphorylation site-specific antibody to the intensity probed with anti-Hsp27. Data with ± SD represents the average of triplicate experiments. C: control; SB: inhibitor SB203580. For the control of HRG-treated cells, untreated cells were cultured for 10 and 30 min and equal amounts of cellular proteins from both time intervals were mixed and used as control. For the control of inhibitor-treated cells, cells were treated with DMSO for 10 hours and cellular proteins were used as the control. * p < 0.05 (student t -test)

Article Snippet: The human BT474 breast cancer cell line was obtained from American Type Culture Collection, and maintained in modified Dulbecco's medium (HybriCare) supplemented with 10% fetal bovine serum (FBS) at 37°C in a humidified atmosphere of 5% CO 2 in an incubator.

Techniques: SDS Page, Cell Culture

Journal: BMC Cancer

Article Title: CD44 isoforms are heterogeneously expressed in breast cancer and correlate with tumor subtypes and cancer stem cell markers

doi: 10.1186/1471-2407-11-418

Figure Lengend Snippet: Comparison of the proportion of CD44+ cells as determined by flow cytometry to the relative mRNA expression determined by q-RT-PCR.

Article Snippet: The breast cell lines BT-474, HCC1428, HCC1937, MCF7, MCF10A, MDA-MB-231, MDA-MB-361, MDA-MB-436, SK-BR-3 and ZR-75-1 were obtained from American Type Culture Collection (ATCC, Mannanas, VA).

Techniques: Flow Cytometry, Expressing

Journal: BMC Cancer

Article Title: OSU-03012 sensitizes breast cancers to lapatinib-induced cell killing: a role for Nck1 but not Nck2

doi: 10.1186/1471-2407-13-256

Figure Lengend Snippet: Isobologram analysis of MDA-MB-231 and BT474 cell lines indicates that the drugs are synergistic in multiple breast cancer lines

Article Snippet: The MDA-MB-231 cell line (purchased from American Type Culture Collection, ATCC) and the BT474 cell line (ATCC) were maintained in RPMI (Invitrogen).

Techniques:

Journal: BMC Cancer

Article Title: OSU-03012 sensitizes breast cancers to lapatinib-induced cell killing: a role for Nck1 but not Nck2

doi: 10.1186/1471-2407-13-256

Figure Lengend Snippet: Phosphorylation of eIF2-α indicates ER stress signaling in MDA-MB-231 and BT474 cells after treatment with OSU-03012 and lapatinib. MDA-MB-231 cells and BT474 cells (1 x 10 6 ) were subjected to vehicle (DMSO, Ctr), OSU-03012 (2 μM), lapatinib (2 μM) or the combination as indicated for 3 h. Cells were then lysed and protein extracts (10-20 μg) were subjected to SDS PAGE followed by western immunoblotting for the indicated proteins.

Article Snippet: The MDA-MB-231 cell line (purchased from American Type Culture Collection, ATCC) and the BT474 cell line (ATCC) were maintained in RPMI (Invitrogen).

Techniques: SDS Page, Western Blot

Journal: BMC Cancer

Article Title: OSU-03012 sensitizes breast cancers to lapatinib-induced cell killing: a role for Nck1 but not Nck2

doi: 10.1186/1471-2407-13-256

Figure Lengend Snippet: The role of eIF2-α phosphorylation in cell death induced by OSU-03012 and lapatinib. A-B : MDA-MB-231 ( A ) or BT474 ( B ) cells (5 x 10 5 ) were transfected with the indicated siRNA molecules and incubated for 48 h. Cells were then treated with either vehicle (Veh) or the combination of OSU-03012 (2 μM) and lapatinib (2 μM) (combo) as indicated and either subjected immunoassays (bottom panels) or plated for clonogenic capacity (top graphs). Numbers indicated are percent control (e.g. Veh). C-D : MDA-MB-231 cells (5 x 10 5 ) were transfected with a control vector (Vector), wild-type (WT) or the Ser 51 Ala mutant (S51A) eIF2-α plasmids. After a further 24 h cells were plated onto 6-well plates to assay for clonogenic capacity ( D ) or subjected to immunoblotting as described in Materials and Methods ( C ). Cells were treated with either vehicle (Veh, DMSO) or OSU-03012 (2 μM) and lapatinib (2 μM) in combination (combo) for 24 h ( D ) or 3 h ( C ). Colonies were allowed to develop for the next 10-14 days. * indicates a p < 0.05 with respect to vehicle-treated cells. E : MDA-MB-231 cells (1 x 10 6 ) were plated and treated with the indicated drugs (Vehicle (Ctr, DMSO), OSU-03012 (OSU, 2 μM), lapatinib (Lap, 2 μM) or the combination (Combo)). Three hours later, cells were harvested and subjected to immunoblotting. Samples were probed with the indicated antibodies (see Materials and Methods).

Article Snippet: The MDA-MB-231 cell line (purchased from American Type Culture Collection, ATCC) and the BT474 cell line (ATCC) were maintained in RPMI (Invitrogen).

Techniques: Transfection, Incubation, Plasmid Preparation, Mutagenesis, Western Blot

Journal: BMC Cancer

Article Title: OSU-03012 sensitizes breast cancers to lapatinib-induced cell killing: a role for Nck1 but not Nck2

doi: 10.1186/1471-2407-13-256

Figure Lengend Snippet: Upstream signaling responsible for eIF2-α phosphorylation: A role for Nck1. A-B : MDA-MB-231 cells and BT474 cells were treated with vehicle (Ctr, DMSO), OSU-03012 (OSU, 2 μM), lapatinib (Lap, 2 μM) or the combination (Combo) for 3 h, then harvested for immunoblotting assays as described in Materials and Methods. Membranes were probed with the indicated antibodies. C : MDA-MB-231 cells (5 x 10 5 ) were transfected with plasmids to express either GFP-Nck1 or GFP-Nck2 as described. After an additional 24 h, cells were treated with the combination of OSU-03012 and lapatinib as indicated for 24 h (upper graphs) or 3 h (lower panels), and then either plated onto 6-well dishes and allowed to form colonies (graphs represent percent control) or harvested for immunoblotting assays and probed with the indicated antibodies. * indicates a p<0.05.

Article Snippet: The MDA-MB-231 cell line (purchased from American Type Culture Collection, ATCC) and the BT474 cell line (ATCC) were maintained in RPMI (Invitrogen).

Techniques: Western Blot, Transfection

Journal: BMC Cancer

Article Title: OSU-03012 sensitizes breast cancers to lapatinib-induced cell killing: a role for Nck1 but not Nck2

doi: 10.1186/1471-2407-13-256

Figure Lengend Snippet: Nck1, but not Nck2 expression ablates the increase in cell death induced by OSU-03012 and lapatinib. A-D : MDA-MB-231 cells ( A, B ), or BT474 cells ( C, D ) (5 x 10 5 ) were co-transfected with either wild-type eIF2-α and the two Nck isoforms ( A, C ), or the S 51 A phospho-mutant and the two Nck isoforms ( B, D ). Cells were allowed to incubate for 24 h to induce ectopic expression, and then treated with either vehicle (indicated with a -), or the combination of OSU-03012 and lapatinib (2 μM each, indicated with a +). Cells were either harvested after a 3 h treatment for western blotting as described in Materials and Methods (bottom panels), or plated singly onto 6-well plates to assay for clonogenic capacity (upper graphs). Cells were treated with OSU-03012 (2 μM) and lapatinib (2 μM) for 24 h, and then allowed to form colonies for 10-14 days. Total colony counts were graphed. * denotes a p-value of <0.05.

Article Snippet: The MDA-MB-231 cell line (purchased from American Type Culture Collection, ATCC) and the BT474 cell line (ATCC) were maintained in RPMI (Invitrogen).

Techniques: Expressing, Transfection, Mutagenesis, Western Blot

Journal: BMC Cancer

Article Title: OSU-03012 sensitizes breast cancers to lapatinib-induced cell killing: a role for Nck1 but not Nck2

doi: 10.1186/1471-2407-13-256

Figure Lengend Snippet: Nck1 is upstream of eIF2-α phosphorylation in the cell death induced by the combination of OSU-03012 and lapatinib. A : BT474 cells (5 x 10 5 ) were co-transfected with the Ser 51 Ala eIF2-α mutant and GFP-Nck1. Cells were allowed to incubate for 24 h to induce ectopic expression then treated with either vehicle (indicated with a -), or the combination of OSU-03012 and lapatinib (2 μM each, indicated with a +). Cells were either harvested for western blotting as described previously (lower panel), or plated into 6-well plates to assay for clonogenic capacity as described previously (upper graph). Graphs represent total colony number. * denotes a p-value of <0.05. B : BT474 (upper panel) and MDA-MB-231 (lower panel) cells were treated with either vehicle (Veh) or OSU-03012 and lapatinib in combination (combo). PP1 was immunoprecipitated and the resulting membranes were immunoblotted with eIF2α and PP1. C : Our data indicate that Nck1 and PP1, which are originally in a complex with eIF2 are dissociated after treatment with the combination of OSU-03012 and lapatinib. This event frees eIF2-α to become phosphorylated by one of many upstream kinases such as PERK, leading to ER stress and eventual cell death.

Article Snippet: The MDA-MB-231 cell line (purchased from American Type Culture Collection, ATCC) and the BT474 cell line (ATCC) were maintained in RPMI (Invitrogen).

Techniques: Transfection, Mutagenesis, Expressing, Western Blot, Immunoprecipitation