Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: HER2 Reactivation through Acquisition of the HER2 L755S Mutation as a Mechanism of Acquired Resistance to HER2-targeted Therapy in HER2+ Breast Cancer

doi: 10.1158/1078-0432.CCR-16-2191

Figure Lengend Snippet: A, Western blot analyses of L/LT-treated BT474 parental and resistant cells. BT474/AZ-P were treated 6h with DMSO or 1μM L. BT474/ATCC-P cells were treated with DMSO or 1μM L + 10μg/ml T. BT474/AZ-LR cells were cultured in the presence of 1μM L and BT474/ATCC-LTR cells were cultured in the presence of 1μM L+ 10μg/ml T. B, Number of mutations detected using whole exome sequencing identified in single samples or multiple samples in the BT474/AZ-P, BT474/AZ-LR, and BT474/ATCC-LTR lines using the BT474/ATCC-P line as reference. C, Pathogenic mutations identified by whole-exome sequencing (supplementary methods) in the BT474/AZ-P, BT474/AZ-LR, and BT474/ATCC-LTR lines using the BT474/ATCC-P line as reference are shown. The shade of blue in each block represents the cancer cell fraction (CCF) of each mutation in each line. Orange squares represent mutations that were found to be expressed using RNA sequencing. Mutations associated with the loss of the wild-type allele are indicated by a diagonal bar. The HER2 (encoded by the gene ERBB2) L755S mutation is highlighted in red.

Article Snippet: Total RNA of the BT474/AZ-P, BT474/AZ-LR, BT474/ATCC-P, and BT474/ATCC-LTR cell lines was extracted with RNeasy Mini Kit (Qiagen).

Techniques: Western Blot, Cell Culture, Sequencing, Blocking Assay, Mutagenesis, RNA Sequencing Assay

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: HER2 Reactivation through Acquisition of the HER2 L755S Mutation as a Mechanism of Acquired Resistance to HER2-targeted Therapy in HER2+ Breast Cancer

doi: 10.1158/1078-0432.CCR-16-2191

Figure Lengend Snippet: A, Dox-inducible ectopic expression of C-terminal HA-tagged WT and mutant HER2 constructs were validated by western blot. B, WT-, G572V-, L755S- and G572V/L755S-HER2-expressing BT474/AZ-P cells were selected with Dox+L for 5 weeks. Pictures were taken at 4X magnification using an Olympus IX70 microscope with a RETIGA 1300R Fast 1394 camera and analyzed with Image-pro plus software (version 5.0). Scale Bar: 50μ. C, WT-, G572V-, L755S-, and G572V/L755S-HER2-expressing BT474/AZ-P cells were treated with or without 1μM L for 6h followed by western blot. L755S- and G572V/L755S-HER2-expressing BT474/AZ-P cells which survived the Dox+L selection for >5 weeks (L755S-LR and G572V/L755S-LR) were analyzed by western blot for HER2 and downstream signaling. D, BT474 parental and HER2-reactivated LR/LTR cells were transfected with siRNA (seq1) targeting the HER2L755S mutant. Culture medium was replaced the next day with regular medium or drug-containing medium, and replaced again at 4 days. Cell growth was assessed at six days by methylene blue assay. Relative percent (%) growth was normalized to mock transfection. Statistical analyses were performed for AZ and ATCC separately. Model–estimated group means and 95% confidence limits were plotted combining two independent experiments.

Article Snippet: Total RNA of the BT474/AZ-P, BT474/AZ-LR, BT474/ATCC-P, and BT474/ATCC-LTR cell lines was extracted with RNeasy Mini Kit (Qiagen).

Techniques: Expressing, Mutagenesis, Construct, Western Blot, Microscopy, Software, Selection, Transfection

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: HER2 Reactivation through Acquisition of the HER2 L755S Mutation as a Mechanism of Acquired Resistance to HER2-targeted Therapy in HER2+ Breast Cancer

doi: 10.1158/1078-0432.CCR-16-2191

Figure Lengend Snippet: A, Responses of BT474/AZ-LR and BT474/ATCC-LTR lines and their relative parental lines (BT474/AZ-P and BT474/ATCC-P) to lapatinib (L), afatinib (Afa), and neratinib (Nrb) were measured by cell growth assay. Data was analyzed by GraphPad Prism (version 6.05) to generate drug response curves and relative IC50 values using the Log (inhibitor) vs. response-variable slope model (Bars=SEM) with normalization of data defining the biggest number in each dataset as 100% and the smallest number in the same dataset as 0%. B, BT474/AZ-P, BT474/AZ-LR, BT474/ATCC-P, and BT474/ATCC-LTR lines were treated with or without 6h of 50nM Afa or Nrb followed by Western blot.

Article Snippet: Total RNA of the BT474/AZ-P, BT474/AZ-LR, BT474/ATCC-P, and BT474/ATCC-LTR cell lines was extracted with RNeasy Mini Kit (Qiagen).

Techniques: Growth Assay, Western Blot

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: HER2 Reactivation through Acquisition of the HER2 L755S Mutation as a Mechanism of Acquired Resistance to HER2-targeted Therapy in HER2+ Breast Cancer

doi: 10.1158/1078-0432.CCR-16-2191

Figure Lengend Snippet: A, Mice prepped with estrogen (E2) pellets were injected with 5×106 BT474/AZ-LR cells and treated with L until randomization to 5 groups: E2+L, E2+Afa, ED+Veh, ED+L, and ED+Afa (for details see Materials and Methods). B, Kaplan-Meier analyses of progression-free survival within 16 days of treatment of E2+Afa or E2+L. Tumor progression was defined as tumor size tripling since randomization. TTP: time to tumor progression. C, Kaplan-Meier analyses of progression-free survival and tumor regression (graph showing change of proportion of non-regressing tumors) within 85 days of ED+Veh, ED+L, or ED+Afa treatment. Tumor regression was defined as tumor size halving since the day of randomization. TTR: time to tumor regression. D, p-HER2 (Y1221), p-AKT (S473), and p-p44/42 MAPK (T202/Y204) levels of xenografts in each treatment arm was assessed by IHC and scored as H-score by a pathologist. Two tumors (Suppl. Fig. S8B and S8C) that were resistant to the ED+Afa regimen were not included in the analysis. *: p<0.05, **: p<0.01, ***: p<0.001.

Article Snippet: Total RNA of the BT474/AZ-P, BT474/AZ-LR, BT474/ATCC-P, and BT474/ATCC-LTR cell lines was extracted with RNeasy Mini Kit (Qiagen).

Techniques: Injection

Journal: Frontiers in Molecular Neuroscience

Article Title: Enhancement of Astroglial Aerobic Glycolysis by Extracellular Lactate-Mediated Increase in cAMP

doi: 10.3389/fnmol.2018.00148

Figure Lengend Snippet: Inhibition of AC reduces the L -lactate- and 3Cl-5OH-BA-induced increase in [cAMP] i and [lactate] i (A,B , panels i) Mean time-courses of FRET ratio signal changes normalized to the maximum signal change for (A,i) Epac1-camps upon addition of 20 mM L -lactate and (B,i) Laconic upon addition of 0.5 mM 3Cl-5OH-BA (black lines) in the absence (white circles) and presence (black circles) of 100 μM DDA, an inhibitor of AC. Each data point represents mean ± s.e.m. ( A,B , panels ii) Mean relative changes in FRET ratio (Rel. ΔFRET ratio) upon L -lactate (A) and (B) 3Cl-5OH-BA stimulation in the absence and presence of DDA. Relative ΔFRET values (%) were calculated by dividing individual ΔFRET values with the average ΔFRET value upon L -lactate or 3Cl-5OH-BA stimulation. Note that the inhibition of AC by DDA causes ∼50 % reduction in L -lactate-induced increase in [cAMP] i in astrocytes and a ∼30–60% reduction in 3Cl-5OH-BA-induced increase in [lactate] i in astrocytes, 3T3-L1 and BT474 cells. In BT474 cells the application of 3Cl-5OH-BA initiated a transient reduction in [lactate] i that was diminished in the presence of DDA. Numbers adjacent to black bars represent number of cells. Data are in the format of the mean ± s.e.m ( ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001). Data for every set of experiment was acquired from at least two different animals.

Article Snippet: BT474 cancer cells (BT-474 Clone 5; ATCC ® CRL-3247 TM ; ATCC-LGC Standards) were grown in Hybri-Care medium (ATCC ® 46-X TM ; ATCC-LGC Standards) supplemented with 1.5 g/L sodium bicarbonate and 10% fetal bovine serum.

Techniques: Inhibition

Journal: Cell reports

Article Title: Molecular Stressors Engender Protein Connectivity Dysfunction through Aberrant N -Glycosylation of a Chaperone

doi: 10.1016/j.celrep.2020.107840

Figure Lengend Snippet: (A and B) Native-PAGE (top) and SDS-PAGE (bottom) separation followed by immunoblot with the 9G10 anti-GRP94 antibody in un-treated cell lines (A) or in those treated for 4 h with PU-WS13 (10 mM) (B). Each data point is an individual cell line; lanes 1–11 are BT474, MDA-MB-468, SKBr3, AU565, MDA-MB-361, HCC1806,MCF7, MDA-MB-231, T47D, BT20, and HMEC. Graph, mean. Error bar, SEM; unpaired t test, **p < 0.01, ***p < 0.001.

Article Snippet: Human: BT-474 , ATCC , Cat# HTB-20, RRID:CVCL_0179.

Techniques: Clear Native PAGE, SDS Page, Western Blot

Journal: Cell reports

Article Title: Molecular Stressors Engender Protein Connectivity Dysfunction through Aberrant N -Glycosylation of a Chaperone

doi: 10.1016/j.celrep.2020.107840

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Human: BT-474 , ATCC , Cat# HTB-20, RRID:CVCL_0179.

Techniques: Recombinant, Isolation, Cell Fractionation, Viability Assay, Transfection, Software, Western Blot, Staining

Journal: Molecular Systems Biology

Article Title: Multi‐modal meta‐analysis of cancer cell line omics profiles identifies ECHDC1 as a novel breast tumor suppressor

doi: 10.15252/msb.20209526

Figure Lengend Snippet: The CLIP signature of ECHDC1 suggests that it was hypermethylated and down‐expressed in all the breast cancer cell lines ( n = 24) in which it was identified as rCCS gene. Breast cancer‐specific survival (BCSS) based on gene expression and methylation levels of ECHDC1 in breast cancer patient tumors in the combined Metabric and Oslo datasets ( n = 3,885). Patients in the low GEXP category class have lower BCSS than those in the non‐low GEXP group. Numbers above the x ‐axis line indicate the number of patients in each group, defined by the color code, at each time point. P ‐value from age‐adjusted Cox‐proportion hazard model. Benign breast epithelial MCF10A and breast carcinoma BT‐474 cells were embedded in 3D collagen as single cells or as spheroids, respectively, and the growth was followed for 5 days. Light micrographs show filamentous actin (phalloidin) and nuclei (Hoechst) in representative cell colonies. Quantitative assessment of the nuclei counts per colony show the induced proliferation in MCF10A cells after ECHCD1 sgRNA knockout. At 72 h, MCF10A mock vs. ECHDC1_sgRNA_1 and ECHDC1_sgRNA_2 P < 0.05; at 96 h mock vs. ECHDC1_sgRNA_1, ECHDC1_sgRNA_2 and ECHDC1_sgRNA_3 P < 0.001; at 120 h mock vs. ECHDC1_sgRNA_1, ECHDC1_sgRNA_2, and ECHDC1_sgRNA_3 P < 0.0001. Nuclei count relative to mock 0 h. Error bars indicate mean ± SEM; n ≥ 10 colonies. Statistical significance was assessed with one‐way ANOVA with Tukey’s multiple comparison test. Scale bar 50 µm. Metabolic pathway of propanoate metabolism. Measured metabolite levels of intermediates in propanoate metabolism in select breast cancer cell lines with or without the ECHDC1 rCCS status ( n = 7 in both groups). Boxes represent the interquartile range, whiskers represent the range of the values and solid line within the box correspond to the median value. Outlier points indicates values not included between the whiskers. Statistical significance was assessed with Wilcoxon test.

Article Snippet: Immortalized breast epithelial cell line MCF10A and breast carcinoma cell line BT‐474 (both American Type Culture Collection; ATCC) were cultured according to manufacturer’s instructions in a humidified incubator with 5% CO 2 at 37°C and routinely checked and tested negative for mycoplasma contamination using MycoAlertPlus™ Mycoplasma Detection Kit (Lonza) according to manufacturer’s instructions.

Techniques: Expressing, Methylation, Knock-Out

Journal: Molecular Systems Biology

Article Title: Multi‐modal meta‐analysis of cancer cell line omics profiles identifies ECHDC1 as a novel breast tumor suppressor

doi: 10.15252/msb.20209526

Figure Lengend Snippet:

Article Snippet: Immortalized breast epithelial cell line MCF10A and breast carcinoma cell line BT‐474 (both American Type Culture Collection; ATCC) were cultured according to manufacturer’s instructions in a humidified incubator with 5% CO 2 at 37°C and routinely checked and tested negative for mycoplasma contamination using MycoAlertPlus™ Mycoplasma Detection Kit (Lonza) according to manufacturer’s instructions.

Techniques: Recombinant, Sequencing, Software

Journal: International Journal of Oncology

Article Title: The anticancer agent YC-1 suppresses progestin-stimulated VEGF in breast cancer cells and arrests breast tumor development

doi: 10.3892/ijo.2012.1675

Figure Lengend Snippet: YC-1 inhibits cell viability and progestin-induced VEGF secretion in human breast cancer cells (n=6 for each concentration). (A), T47-D and BT-474 cells were treated with indicated concentrations of YC-1 for 18 h and cell viability was determined using the SRB assay. * Significantly different from control (p<0.001, ANOVA). (B), T47-D cells were pre-treated for 30 min with 1 μ M RU-486, or 100 μ M YC-1 and then treated with 10 nM progesterone or MPA or with synthetic progestins (C). Media was collected after 16–18 h and analyzed for VEGF using ELISA as described in Materials and methods (n=3–12). One-way ANOVA was used to statistically analyze data. * Significant induction compared with control. ** Significant inhibition compared with appropriate progestin control, p<0.05. The control value for VEGF in represents 496±51 pg/mg cellular protein.

Article Snippet: Human T47-D and BT-474 breast cancer cell lines were obtained from ATCC (Manassas, VA).

Techniques: Concentration Assay, Sulforhodamine B Assay, Enzyme-linked Immunosorbent Assay, Inhibition