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: iScience

Article Title: AMPK is required for recovery from metabolic stress induced by ultrasound microbubble treatment

doi: 10.1016/j.isci.2022.105883

Figure Lengend Snippet: Design of the study (A) MDA-MB-231, SUM149PT or BT-20 cells were prepared and treated with USMB using a calibrated acoustic exposure platform that was developed to deliver ultrasound pulses, as was previously described. See for additional details of USMB treatment. Numerous assays were performed following USMB treatment over a 72 h period as indicated, including metabolite analysis ( Figures 2 and ), Signaling Analysis ( Figures 3 and ) and proliferation and viability assays ( Figures 6 and ). (B) MDA-MB-231 cells were subject to USMB treatment (USMB) or 2 μg/mL digitonin for 5 min in the continuous presence of Lucifer Yellow Potassium Salt (125 μg/mL), as indicated, to label (reversibly or irreversibly) sonoporated cells. Shown (left panels) are representative images obtained by widefield epifluorescence microscopy, scale bar = 100 μm. Also shown (right panels) is the quantification of Lucifer Yellow fluorescence, depicting fluorescence measured in individual cells (individual points) and median (horizontal bar) with 95% confidence interval (CI). (C) MDA-MB-231 cells were treated with USMB and then only subsequently incubated with Propidium Iodide (PI), which thus identifies irreversibly sonoporated cells. For some cell monolayer samples, no wash was performed after USMB treatment (USMB no wash), whereas the USMB + wash condition involved several washes to remove non-viable cells before PI staining and imaging; the latter is the standard experimental protocol used throughout this study. Shown (left panels) are representative images as overlays of phase contrast and PI fluorescence. Scale bar, 400 μm. Also shown (right panel) is the quantification of PI-positive cells, showing the mean ± SD of 2 independent experiments.

Article Snippet: The human breast cancer cell line BT-20 was obtained from AmericanType Culture Collection, ATCC (HTB-19).

Techniques: Epifluorescence Microscopy, Fluorescence, Incubation, Staining, Imaging

Journal: iScience

Article Title: AMPK is required for recovery from metabolic stress induced by ultrasound microbubble treatment

doi: 10.1016/j.isci.2022.105883

Figure Lengend Snippet: USMB triggers AMPK activation (A–C) MDA-MB-231, SUM149PT, and BT-20 cells were treated with USMB or left untreated (basal). Whole cell lysates were prepared 30 min after USMB treatment and were analyzed by Western blotting with depicted antibodies. (D and E) MDA-MB-231 cells were fixed, permeabilized, and stained for endogenous pS79-ACC, 30 min after USMB treatment. Some samples were also pre-treated with 10 μM compound C for 1 h before USMB treatment, as indicated. Shown in (D) are representative images obtained by widefield epifluorescence microscopy, scale bar, 100 μm. Shown in (E) is the quantification of immunofluorescence intensity; data is represented as normalized fluorescence (relative units, R.U.) values in individual cells (small circles), average cell fluorescence intensity in each experiment in each condition (large shapes) and mean of independent experiments (bar) ± SD (whiskers). The experiment was repeated three times independently. Measurements are color-coded by independent experiment. Statistical analysis was performed by one-way ANOVA followed by Tukey’s multiple comparisons test. ∗, p < 0.05. (F) A similar experiment was performed in which MDA-MB-231 were subject to USMB in the presence of Lucifer Yellow Potassium Salt (125 μg/mL), then subject to fixation and staining. Shown is the quantification of immunofluorescence intensity of phospho-ACC and Lucifer Yellow for individual MDA-MB-231 cells treated with USMB as indicated.

Article Snippet: The human breast cancer cell line BT-20 was obtained from AmericanType Culture Collection, ATCC (HTB-19).

Techniques: Activation Assay, Western Blot, Staining, Epifluorescence Microscopy, Immunofluorescence, Fluorescence

Journal: Molecular Cancer

Article Title: DNMT3b overexpression contributes to a hypermethylator phenotype in human breast cancer cell lines

doi: 10.1186/1476-4598-7-15

Figure Lengend Snippet: Methylation analysis of CST6, LCN2, SCNN1A, CDH1, CEACAM6 , and ESR1 among putative hypermethylator and low-frequency methylator cell lines . (A) Representative agarose gels of methylation-specific PCR (MSP) products corresponding to CST6, LCN2, SCNN1A, CDH1, CEACAM6 , and ESR1 are shown. U = unmethylated MSP product, M = methylated MSP product. Cell line abbreviations are as follows: 231 = MDA-MB-231, 415 = MDA-MB-415, 435S = MDA-MB-435S, 436 = MDA-MB-436, 453 = MDA-MB-453, and 468 = MDA-MB-468. All other cell lines are designated by their full name. (B) Representative bisulfite sequence analysis for CDH1 . Methylated CpGs are designated by closed circles, unmethylated CpGs are designated by open circles for MDA-MB-435S, BT20, and MDA-MB-231 cell lines (5 replicates each). (C) Representative agarose gels of RT-PCR products for CST6, SCNN1A, CDH1, CEACAM6 , and ESR1 demonstrating 5-aza induction of gene expression in hypermethylator cell lines. RT-PCR results using cDNA template from untreated (-) and 5-aza treated (+) are shown.

Article Snippet: Human breast cancer cell lines BT20 (ATCC# HTB19), BT549 (HTB122), Hs578T (HTB126), MCF7 (HTB22), MDA-MB-231 (HTB26), MDA-MB-415 (HTB128), MDA-MB-435S (HTB129), MDA-MB-436 (HTB130), MDA-MB-453 (HTB131), MDA-MB-468 (HTB132), SKBR3 (HTB30), and ZR-75-1 (CRL-1500) were obtained from the Tissue Culture Core Facility of the University of North Carolina Lineberger Comprehensive Cancer Center (Chapel Hill, NC), and the normal breast epithelial cell line MCF12A [ ] (CRL-10782) was obtained from the American Type Culture Collection [ ].

Techniques: Methylation, Sequencing, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: Breast Cancer Research : BCR

Article Title: β1 integrin mediates an alternative survival pathway in breast cancer cells resistant to lapatinib

doi: 10.1186/bcr2936

Figure Lengend Snippet: Phosphorylated levels of β1 downstream kinases are increased upon acquisition of resistance to lapatinib (L) . ( A ) Parental (P) BT474 and ( B ) HCC1954 cells resistant to lapatinib (LRes), trastuzumab (TRes), and combination (LTRes) treatment strategies were developed by long-term exposure in 2D. Protein extracts were probed for β1, pHER2, pFAK, and pSrc, as well as totals.

Article Snippet: The human breast cancer cell line BT474 was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained as previously described [ ].

Techniques:

Journal: Breast Cancer Research : BCR

Article Title: β1 integrin mediates an alternative survival pathway in breast cancer cells resistant to lapatinib

doi: 10.1186/bcr2936

Figure Lengend Snippet: β1 blockade overcomes lapatinib resistance in BT474 cells in 3D culture . ( A ) Parental and LRes cells were plated on lrECM and treated ± lapatinib. ( B ) The β1 inhibitory antibody AIIB2--or IgG control--was applied to parental and LRes cells on Day 0 of plating on lrECM and allowed to grow for 12 days. ( C ) Parental and LRes cells were plated in lrECM with appropriate inhibitors, allowed to grow for five days, stained with Ki67 antigen or TUNEL labeling, and imaged. ( D ) 3D cultures of BT474 parental and LRes cells treated with AIIB2 or IgG were allowed to propagate for five days, then harvested for protein and immunoblotting. Densitometry measurements were normalized to total levels--except β1, which was normalized to β-actin--and are representative of three independent experiments. ( E ) The FAK inhibitor PF 573228 was applied to parental and LRes cells on Day 0 of plating on lrECM and cultures were allowed to grow for 12 days.

Article Snippet: The human breast cancer cell line BT474 was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained as previously described [ ].

Techniques: Staining, TUNEL Assay, Labeling, Western Blot

Journal: Breast Cancer Research : BCR

Article Title: β1 integrin mediates an alternative survival pathway in breast cancer cells resistant to lapatinib

doi: 10.1186/bcr2936

Figure Lengend Snippet: Percent growth inhibition of cells in response to HER-targeted therapies

Article Snippet: The human breast cancer cell line BT474 was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained as previously described [ ].

Techniques: Inhibition

Journal: Breast Cancer Research : BCR

Article Title: β1 integrin mediates an alternative survival pathway in breast cancer cells resistant to lapatinib

doi: 10.1186/bcr2936

Figure Lengend Snippet: HER2 and the β1 pathway play alternate roles in resistance to lapatinib-containing regimens, in comparison to trastuzumab . ( A ) Parental, LRes, and TRes cells were plated on lrECM in the presence of lapatinib and/or trastuzumab and assayed for response. ( B ) 3D cultures of parental, LRes, and TRes BT474 cells were harvested for protein and probed for phosphorylated and total HER receptors.

Article Snippet: The human breast cancer cell line BT474 was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained as previously described [ ].

Techniques:

Journal: Breast Cancer Research : BCR

Article Title: β1 integrin mediates an alternative survival pathway in breast cancer cells resistant to lapatinib

doi: 10.1186/bcr2936

Figure Lengend Snippet: β1 inhibition impedes colony grow of BT474 LRes and LTRes cells, but not parental or TRes . ( A ) Parental, LRes, LTRes, and TRes cells were plated in lrECM, subjected to HER2 and/or β1 inhibitors on Day 0, and propagated for 10 to 12 days. ( B ) The Hs_ITGB1_5 siRNA was validated both in 2D and 3D (top), transfected at 40 nMsi into parental, LRes, LTRes, and TRes cells, which were then grown on lrECM for 10 days (bottom).

Article Snippet: The human breast cancer cell line BT474 was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained as previously described [ ].

Techniques: Inhibition, Transfection

Journal: Experimental and Therapeutic Medicine

Article Title: Inhibition of miRNA-34a promotes triple negative cancer cell proliferation by promoting glucose uptake

doi: 10.3892/etm.2019.8017

Figure Lengend Snippet: Effects of miRNA-34a inhibition on glucose uptake and GLUT1 expression in triple negative breast cancer cell lines and a normal breast epithelial tissue cell line. Normalized (A) miRNA-34a inhibition, (B) glucose uptake and (C) GLUT1 expression in triple negative breast cancer cell lines BT-20 and MDA-MB-231 and a normal breast epithelial tissue cell line MCF-12F are presented. *P<0.05. miRNA, microRNA; GLUT1, glucose transporter 1; C, control; NC, negative control.

Article Snippet: Human triple negative breast cancer cell lines BT-20 (ATCC ® HTB-19™) and MDA-MB-231 (ATCC ® HTB-26™), as well as a normal human breast epithelial tissue cell line MCF-12F (ATCC ® CRL-10783™) were purchased from the American Type Culture Collection.

Techniques: Inhibition, Expressing, Negative Control