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: 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: 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: 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: PLoS ONE

Article Title: Down-Regulation of Fil amin A i nteracting p rotein 1 - l ike Is Associated with Promoter Methylation and an Invasive Phenotype in Breast, Colon, Lung and Pancreatic Cancers

doi: 10.1371/journal.pone.0082620

Figure Lengend Snippet: qRT-PCR analysis for FILIP1L conducted on cDNA from BT-549 breast ( A ), HT-29 colon ( B ), H1299 lung ( C ) and MIA PaCa-2 pancreatic ( D ) cancer cell lines treated with either 5-aza-2’-deoxycytidine (DAC) or Trichostatin A (TSA). Values 0.1, 1 or 10 in the x axis indicate the concentration of DAC and TSA in μM. The y axis represents fold change of each reagent-treated cell type over DMSO-treated control cells, where each value was standardized with the housekeeping gene hRPL7 . Error bars indicate SEM ( n = 3). The result is an average of two independent experiments. P values are derived from comparison between DMSO-treated control and either DAC- or TSA-treated experiments. NS indicates not significant.

Article Snippet: Human pancreatic cancer cell lines MIA PaCa-2, PANC-1, Hs 766T, HPAC, HPAF-II, SU.86.86, Panc 02.03 and Capan-1 and human breast cancer cell lines BT-549, Hs 578T, MDA-MB-468, BT-474 and ZR-75-1 were purchased from ATCC.

Techniques: Quantitative RT-PCR, Concentration Assay, Derivative Assay

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