human breast carcinoma cell lines  (ATCC)


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    ATCC human breast carcinoma cell lines
    Human Breast Carcinoma Cell Lines, supplied by ATCC, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    bt 20  (ATCC)


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    ATCC bt 20
    Bt 20, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    bt 474 cells cat htb 20 cells  (ATCC)


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    ATCC bt 474 cells cat htb 20 cells
    MCF-7 and <t>BT-474</t> cells were cultured on soft (0.5 kPa) or stiff (9 kPa) supports for 2 weeks before subsequent analysis. a Docetaxel (Doc) induced apoptosis of MCF-7 and BT-474 was assessed by flow cytometry. The proportions of Annexin V + cells are shown. b Quantitation of apoptotic cells (Annexin V + ) in MCF-7 and BT-474 cells in (a) ( n = 5 biologically independent experiments). Mean ± SD, **** p < 0.0001 by two-sided two-way ANOVA with Tukey test. c MCF-7 and BT-474 cells cultured on soft or stiff supports were treated with docetaxel or cisplatin. Cell viability was evaluated after 24 h by CCK8 assays ( n = 5 biologically independent experiments). Mean ± SD, **** p < 0.0001 by two-sided Student’s t test. d Western blots for cleaved/uncleaved caspase-3 and PARP in docetaxel-treated MCF-7 and BT-474 cells cultured on soft or stiff supports. Representative images from 3 biologically independent experiments are shown. e The percentage of CD44 high CD24 low cells in MCF-7 and BT-474 cells cultured on soft or stiff supports were determined by flow cytometry. f Quantitation of CD44 high CD24 low cells in (e) ( n = 5 biologically independent experiments). Mean ± SD, **** p < 0.0001 by two-sided Student’s t test. For b-d and f , source data are provided as a Source Data file.
    Bt 474 Cells Cat Htb 20 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Niche stiffness sustains cancer stemness via TAZ and NANOG phase separation"

    Article Title: Niche stiffness sustains cancer stemness via TAZ and NANOG phase separation

    Journal: Nature Communications

    doi: 10.1038/s41467-023-35856-y

    MCF-7 and BT-474 cells were cultured on soft (0.5 kPa) or stiff (9 kPa) supports for 2 weeks before subsequent analysis. a Docetaxel (Doc) induced apoptosis of MCF-7 and BT-474 was assessed by flow cytometry. The proportions of Annexin V + cells are shown. b Quantitation of apoptotic cells (Annexin V + ) in MCF-7 and BT-474 cells in (a) ( n = 5 biologically independent experiments). Mean ± SD, **** p < 0.0001 by two-sided two-way ANOVA with Tukey test. c MCF-7 and BT-474 cells cultured on soft or stiff supports were treated with docetaxel or cisplatin. Cell viability was evaluated after 24 h by CCK8 assays ( n = 5 biologically independent experiments). Mean ± SD, **** p < 0.0001 by two-sided Student’s t test. d Western blots for cleaved/uncleaved caspase-3 and PARP in docetaxel-treated MCF-7 and BT-474 cells cultured on soft or stiff supports. Representative images from 3 biologically independent experiments are shown. e The percentage of CD44 high CD24 low cells in MCF-7 and BT-474 cells cultured on soft or stiff supports were determined by flow cytometry. f Quantitation of CD44 high CD24 low cells in (e) ( n = 5 biologically independent experiments). Mean ± SD, **** p < 0.0001 by two-sided Student’s t test. For b-d and f , source data are provided as a Source Data file.
    Figure Legend Snippet: MCF-7 and BT-474 cells were cultured on soft (0.5 kPa) or stiff (9 kPa) supports for 2 weeks before subsequent analysis. a Docetaxel (Doc) induced apoptosis of MCF-7 and BT-474 was assessed by flow cytometry. The proportions of Annexin V + cells are shown. b Quantitation of apoptotic cells (Annexin V + ) in MCF-7 and BT-474 cells in (a) ( n = 5 biologically independent experiments). Mean ± SD, **** p < 0.0001 by two-sided two-way ANOVA with Tukey test. c MCF-7 and BT-474 cells cultured on soft or stiff supports were treated with docetaxel or cisplatin. Cell viability was evaluated after 24 h by CCK8 assays ( n = 5 biologically independent experiments). Mean ± SD, **** p < 0.0001 by two-sided Student’s t test. d Western blots for cleaved/uncleaved caspase-3 and PARP in docetaxel-treated MCF-7 and BT-474 cells cultured on soft or stiff supports. Representative images from 3 biologically independent experiments are shown. e The percentage of CD44 high CD24 low cells in MCF-7 and BT-474 cells cultured on soft or stiff supports were determined by flow cytometry. f Quantitation of CD44 high CD24 low cells in (e) ( n = 5 biologically independent experiments). Mean ± SD, **** p < 0.0001 by two-sided Student’s t test. For b-d and f , source data are provided as a Source Data file.

    Techniques Used: Cell Culture, Flow Cytometry, Quantitation Assay, Western Blot

    MCF-7 and BT-474 cells were cultured on soft or stiff supports for 2 weeks before subsequent analysis. In some experiments, cells were pre-transduced with TAZ shRNA (shTAZ-1 or shTAZ-2) or GFP shRNA (shGFP). a The expression of TAZ in MCF-7 and BT-474 cells cultured on soft or stiff supports was determined by western blot ( n = 3 biologically independent experiments). b Representative immunofluorescence staining of TAZ in MCF-7 and BT-474 cells cultured on soft or stiff substrates. The insert represents a magnification from the indicated area (white square). Scale bars, 10 μm. c Proportion of cancer cells exhibiting mainly nuclear TAZ localization (N > C), diffuse distribution of TAZ in nucleus and cytoplasm (N = C), or preferential cytoplasmic TAZ (N < C or undetectable). 20 cells were assessed per group, experiments were independently repeated 4 times. Mean ± SD, **** p < 0.0001 by two-tailed chi-square test. d Docetaxel induced apoptosis of MCF-7 and BT-474 was assessed by flow cytometry. See Supplementary Fig. for quantification. e MCF-7 and BT-474 cells cultured on soft or stiff supports were treated with docetaxel or cisplatin. Cell viability was evaluated by CCK8 assay ( n = 5 biologically independent experiments). Mean ± SD, **** p < 0.0001 by two-sided one-way ANOVA with Tukey test. f Representative images of CD44 high CD24 low cells in MCF-7 and BT-474 cells were detected by flow cytometry. See Supplementary Fig. for quantification. g Western blots of TAZ, SOX2, and OCT4 in MCF-7 and BT-474 cells cultured on soft or stiff supports ( n = 3 biologically independent experiments). For data presented in the same figure panel, the samples were derived from the same experiment and blots were processed in parallel. For a , c , e and g , source data are provided as a Source Data file.
    Figure Legend Snippet: MCF-7 and BT-474 cells were cultured on soft or stiff supports for 2 weeks before subsequent analysis. In some experiments, cells were pre-transduced with TAZ shRNA (shTAZ-1 or shTAZ-2) or GFP shRNA (shGFP). a The expression of TAZ in MCF-7 and BT-474 cells cultured on soft or stiff supports was determined by western blot ( n = 3 biologically independent experiments). b Representative immunofluorescence staining of TAZ in MCF-7 and BT-474 cells cultured on soft or stiff substrates. The insert represents a magnification from the indicated area (white square). Scale bars, 10 μm. c Proportion of cancer cells exhibiting mainly nuclear TAZ localization (N > C), diffuse distribution of TAZ in nucleus and cytoplasm (N = C), or preferential cytoplasmic TAZ (N < C or undetectable). 20 cells were assessed per group, experiments were independently repeated 4 times. Mean ± SD, **** p < 0.0001 by two-tailed chi-square test. d Docetaxel induced apoptosis of MCF-7 and BT-474 was assessed by flow cytometry. See Supplementary Fig. for quantification. e MCF-7 and BT-474 cells cultured on soft or stiff supports were treated with docetaxel or cisplatin. Cell viability was evaluated by CCK8 assay ( n = 5 biologically independent experiments). Mean ± SD, **** p < 0.0001 by two-sided one-way ANOVA with Tukey test. f Representative images of CD44 high CD24 low cells in MCF-7 and BT-474 cells were detected by flow cytometry. See Supplementary Fig. for quantification. g Western blots of TAZ, SOX2, and OCT4 in MCF-7 and BT-474 cells cultured on soft or stiff supports ( n = 3 biologically independent experiments). For data presented in the same figure panel, the samples were derived from the same experiment and blots were processed in parallel. For a , c , e and g , source data are provided as a Source Data file.

    Techniques Used: Cell Culture, Transduction, shRNA, Expressing, Western Blot, Immunofluorescence, Staining, Two Tailed Test, Flow Cytometry, CCK-8 Assay, Derivative Assay

    a – d TAZ and NANOG were knockout or overexpressed by lentiviral transfection in MCF-7 and BT-474 cells. NANOG, TAZ, SOX2 or/and OCT4 were overexpressed in TAZ or NANOG knockout cells. Transfected cells were cultured on stiff hydrogels for 2 weeks. OE, overexpression. a (Left) Representative flow cytometric plot indicating the percentages of CD44 high CD24 low cells in MCF-7 and BT-474 cells. (Right) Qualification of five biologically independent experiments. **** p < 0.0001; for MCF-7 cells, *** p = 0.0003, ** p = 0.0039, ns=0.9899; for BT-474 cells, *** p = 0.0008, ns=0.9982. b Cell viability of MCF-7 and BT-474 cells treated with various concentrations of docetaxel and cisplatin for 24 h was determined by CCK8 assays ( n = 4 biologically independent experiments). P values compared with sgTAZ + Vector group were shown in the figure. c (Left) Representative flow cytometric plot indicating the percentages of CD44 high CD24 low cells in MCF-7 and BT-474 cells. (Right) Qualification of five independent experiments. **** p < 0.0001; for MCF-7 cells, *** p = 0.0004, ** p = 0.0033, ns=0.9400; for BT-474 cells, *** p = 0.0002, ** p = 0.0013, ns=0.9982. d Cell viability of MCF-7 and BT-474 cells treated with various concentrations of docetaxel and cisplatin for 24 h was determined by CCK8 assays ( n = 4 biologically independent experiments). P values compared with sgNANOG + Vector group were shown in the figure. For panels (a– d) , data are mean ± SD; p values were calculated by two-sided one-way ANOVA with Tukey test. e , f MCF-7 cells were cultured on soft or stiff hydrogels for 2 weeks. Then ChIP was performed using cell lysates with indicated antibodies. e The precipitated DNA using IgG and TAZ antibody, respectively, was quantified by qPCR with primers specific to promoter regions of Sox2 and Pou5f1 . f The precipitated DNA using IgG and NANOG antibody, respectively, was quantified by qPCR with primers specific to promoter regions of Sox2 and Pou5f1 . e , f Fold enrichment was normalized to IgG-ChIP negative control ( n = 4 biologically independent experiments). Numeric values denote mean ± SD; **** p < 0.0001 by two-sided two-way ANOVA with Tukey test. For a–f , source data are provided as a Source Data file.
    Figure Legend Snippet: a – d TAZ and NANOG were knockout or overexpressed by lentiviral transfection in MCF-7 and BT-474 cells. NANOG, TAZ, SOX2 or/and OCT4 were overexpressed in TAZ or NANOG knockout cells. Transfected cells were cultured on stiff hydrogels for 2 weeks. OE, overexpression. a (Left) Representative flow cytometric plot indicating the percentages of CD44 high CD24 low cells in MCF-7 and BT-474 cells. (Right) Qualification of five biologically independent experiments. **** p < 0.0001; for MCF-7 cells, *** p = 0.0003, ** p = 0.0039, ns=0.9899; for BT-474 cells, *** p = 0.0008, ns=0.9982. b Cell viability of MCF-7 and BT-474 cells treated with various concentrations of docetaxel and cisplatin for 24 h was determined by CCK8 assays ( n = 4 biologically independent experiments). P values compared with sgTAZ + Vector group were shown in the figure. c (Left) Representative flow cytometric plot indicating the percentages of CD44 high CD24 low cells in MCF-7 and BT-474 cells. (Right) Qualification of five independent experiments. **** p < 0.0001; for MCF-7 cells, *** p = 0.0004, ** p = 0.0033, ns=0.9400; for BT-474 cells, *** p = 0.0002, ** p = 0.0013, ns=0.9982. d Cell viability of MCF-7 and BT-474 cells treated with various concentrations of docetaxel and cisplatin for 24 h was determined by CCK8 assays ( n = 4 biologically independent experiments). P values compared with sgNANOG + Vector group were shown in the figure. For panels (a– d) , data are mean ± SD; p values were calculated by two-sided one-way ANOVA with Tukey test. e , f MCF-7 cells were cultured on soft or stiff hydrogels for 2 weeks. Then ChIP was performed using cell lysates with indicated antibodies. e The precipitated DNA using IgG and TAZ antibody, respectively, was quantified by qPCR with primers specific to promoter regions of Sox2 and Pou5f1 . f The precipitated DNA using IgG and NANOG antibody, respectively, was quantified by qPCR with primers specific to promoter regions of Sox2 and Pou5f1 . e , f Fold enrichment was normalized to IgG-ChIP negative control ( n = 4 biologically independent experiments). Numeric values denote mean ± SD; **** p < 0.0001 by two-sided two-way ANOVA with Tukey test. For a–f , source data are provided as a Source Data file.

    Techniques Used: Knock-Out, Transfection, Cell Culture, Over Expression, Plasmid Preparation, Negative Control

    breast cancer cell lines bt 20  (ATCC)


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    ATCC breast cancer cell lines bt 20
    SUSD4 expression suppresses tumor growth and affect EGFR dynamics. Tumor growth monitored in a syngeneic mouse model. 4 T1-Luc2 cells stably expressing mouse SUSD4, or mock control cells were injected into the mammary fat pad of BALB/c mice ( n = 10 mice per group). In first experiment ( A ) 2 × 10 6 cells were injected per mouse and in the second ( B ) 5 × 10 6 cells were injected per mouse (Error bars represents standard error of the mean). ( C ) Migration and invasion assay ( D ) of 5 × 10 4 4 T1-Luc2 cells incubated for 48 h under serum. (E) Immunodetection of SUSD4 from lysates of <t>BT-20</t> cells stably expressing human SUSD4 or mock control cells treated with deglycosylation enzymes to assess SUSD4 N-glycosylation status. (F) Protein profile comparison between SUSD4-expressing BT-20 cells and mock control cells using the Human XL Oncology Array ( n = 2 technical repeats). (G) Expression of SUSD4 and EGFR, independent of each other, in individual cell types present in breast cancer tumors. Representative immunoblots depicting total EGFR levels and phospho-EGFR at Tyr1045 (H) or Tyr1086 (I) in SUSD4-expressing BT-20 or MDA-MB-468 cells and corresponding mock control cells. Densitometric analysis of total EGFR and phospho-EGFR levels together with the ratio of phosphorylated EGFR to total EGFR in BT-20 cells (J) and MDA-MB-468 cells (K). (L) EGFR ubiquitination assessed by immunoprecipitation of EGFR in serum-starved EGF-treated BT-20 cell expressing SUSD4 or mock control cells followed by immunoblot detection of ubiquitin. All results were repeated in at least three independent biological experiments unless otherwise specified. The p -value was estimated with t test when two groups were compared and with Two-way ANOVA test when two or more groups were compared for two independent parameters * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001
    Breast Cancer Cell Lines Bt 20, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    breast cancer cell lines bt 20 - by Bioz Stars, 2023-02
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    1) Product Images from "Sushi domain-containing protein 4 binds to epithelial growth factor receptor and initiates autophagy in an EGFR phosphorylation independent manner"

    Article Title: Sushi domain-containing protein 4 binds to epithelial growth factor receptor and initiates autophagy in an EGFR phosphorylation independent manner

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-022-02565-1

    SUSD4 expression suppresses tumor growth and affect EGFR dynamics. Tumor growth monitored in a syngeneic mouse model. 4 T1-Luc2 cells stably expressing mouse SUSD4, or mock control cells were injected into the mammary fat pad of BALB/c mice ( n = 10 mice per group). In first experiment ( A ) 2 × 10 6 cells were injected per mouse and in the second ( B ) 5 × 10 6 cells were injected per mouse (Error bars represents standard error of the mean). ( C ) Migration and invasion assay ( D ) of 5 × 10 4 4 T1-Luc2 cells incubated for 48 h under serum. (E) Immunodetection of SUSD4 from lysates of BT-20 cells stably expressing human SUSD4 or mock control cells treated with deglycosylation enzymes to assess SUSD4 N-glycosylation status. (F) Protein profile comparison between SUSD4-expressing BT-20 cells and mock control cells using the Human XL Oncology Array ( n = 2 technical repeats). (G) Expression of SUSD4 and EGFR, independent of each other, in individual cell types present in breast cancer tumors. Representative immunoblots depicting total EGFR levels and phospho-EGFR at Tyr1045 (H) or Tyr1086 (I) in SUSD4-expressing BT-20 or MDA-MB-468 cells and corresponding mock control cells. Densitometric analysis of total EGFR and phospho-EGFR levels together with the ratio of phosphorylated EGFR to total EGFR in BT-20 cells (J) and MDA-MB-468 cells (K). (L) EGFR ubiquitination assessed by immunoprecipitation of EGFR in serum-starved EGF-treated BT-20 cell expressing SUSD4 or mock control cells followed by immunoblot detection of ubiquitin. All results were repeated in at least three independent biological experiments unless otherwise specified. The p -value was estimated with t test when two groups were compared and with Two-way ANOVA test when two or more groups were compared for two independent parameters * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001
    Figure Legend Snippet: SUSD4 expression suppresses tumor growth and affect EGFR dynamics. Tumor growth monitored in a syngeneic mouse model. 4 T1-Luc2 cells stably expressing mouse SUSD4, or mock control cells were injected into the mammary fat pad of BALB/c mice ( n = 10 mice per group). In first experiment ( A ) 2 × 10 6 cells were injected per mouse and in the second ( B ) 5 × 10 6 cells were injected per mouse (Error bars represents standard error of the mean). ( C ) Migration and invasion assay ( D ) of 5 × 10 4 4 T1-Luc2 cells incubated for 48 h under serum. (E) Immunodetection of SUSD4 from lysates of BT-20 cells stably expressing human SUSD4 or mock control cells treated with deglycosylation enzymes to assess SUSD4 N-glycosylation status. (F) Protein profile comparison between SUSD4-expressing BT-20 cells and mock control cells using the Human XL Oncology Array ( n = 2 technical repeats). (G) Expression of SUSD4 and EGFR, independent of each other, in individual cell types present in breast cancer tumors. Representative immunoblots depicting total EGFR levels and phospho-EGFR at Tyr1045 (H) or Tyr1086 (I) in SUSD4-expressing BT-20 or MDA-MB-468 cells and corresponding mock control cells. Densitometric analysis of total EGFR and phospho-EGFR levels together with the ratio of phosphorylated EGFR to total EGFR in BT-20 cells (J) and MDA-MB-468 cells (K). (L) EGFR ubiquitination assessed by immunoprecipitation of EGFR in serum-starved EGF-treated BT-20 cell expressing SUSD4 or mock control cells followed by immunoblot detection of ubiquitin. All results were repeated in at least three independent biological experiments unless otherwise specified. The p -value was estimated with t test when two groups were compared and with Two-way ANOVA test when two or more groups were compared for two independent parameters * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001

    Techniques Used: Expressing, Stable Transfection, Injection, Migration, Invasion Assay, Incubation, Immunodetection, Western Blot, Immunoprecipitation

    SUSD4 expression leads to increased autophagic flux. Autophagy levels SUSD4-expressing BT-20 cells (A and C) or MDA-MB-468 cells (B and D) and corresponding mock control cells treated with chloroquine for up to 5 hours. Representative immunoblots (A and B) and densitometric analysis (C and D) of LC3B-II levels in SUSD4-expressing cells and mock control cells for each cell line and chloroquine treatment time. Autophagy levels depicted by p62 levels in SUSD4-expressing BT-20 cells (E and G) or MDA-MB-468 cells (F and H) and corresponding mock control cells treated with chloroquine for 2- or 4 hours. Representative immunoblots and densitometric analysis of p62 levels in BT-20 cells (E and G) and MDA-MB-468 cells (F and H) . LC3B puncta in SUSD4-expressing BT-20 cells or MDA-MB-468 cells and corresponding mock control cells expressing GPF-tagged LC3B visualized by confocal microscopy. Representative images showing LC3B puncta (green) and DAPI (blue) (I) as well as quantified number of puncta per cell (J) in untreated cells and cells treated with chloroquine for 3 hours (Every data point represents the average of puncta per cell from at least 10 cells per sample). Assessment of autophagosome-lysosome fusion in BT-20 cells or MDA-MB-468 cell expressing SUSD4 and corresponding mock control cells using the Premo Autophagy Tandem Sensor RFP-GFP-LC3B (K), created with BioRender.com . Representative images (L) and quantified colocalization coefficient (M) of magenta- and green-stained vesicles. (Every data point represents the average of at least 10 cells per sample). All results were repeated in at least three independent biological experiments. The p-value was estimated with t test when two groups were compared and with Two-way ANOVA test when two or more groups were compared for two independent parameters * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001
    Figure Legend Snippet: SUSD4 expression leads to increased autophagic flux. Autophagy levels SUSD4-expressing BT-20 cells (A and C) or MDA-MB-468 cells (B and D) and corresponding mock control cells treated with chloroquine for up to 5 hours. Representative immunoblots (A and B) and densitometric analysis (C and D) of LC3B-II levels in SUSD4-expressing cells and mock control cells for each cell line and chloroquine treatment time. Autophagy levels depicted by p62 levels in SUSD4-expressing BT-20 cells (E and G) or MDA-MB-468 cells (F and H) and corresponding mock control cells treated with chloroquine for 2- or 4 hours. Representative immunoblots and densitometric analysis of p62 levels in BT-20 cells (E and G) and MDA-MB-468 cells (F and H) . LC3B puncta in SUSD4-expressing BT-20 cells or MDA-MB-468 cells and corresponding mock control cells expressing GPF-tagged LC3B visualized by confocal microscopy. Representative images showing LC3B puncta (green) and DAPI (blue) (I) as well as quantified number of puncta per cell (J) in untreated cells and cells treated with chloroquine for 3 hours (Every data point represents the average of puncta per cell from at least 10 cells per sample). Assessment of autophagosome-lysosome fusion in BT-20 cells or MDA-MB-468 cell expressing SUSD4 and corresponding mock control cells using the Premo Autophagy Tandem Sensor RFP-GFP-LC3B (K), created with BioRender.com . Representative images (L) and quantified colocalization coefficient (M) of magenta- and green-stained vesicles. (Every data point represents the average of at least 10 cells per sample). All results were repeated in at least three independent biological experiments. The p-value was estimated with t test when two groups were compared and with Two-way ANOVA test when two or more groups were compared for two independent parameters * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001

    Techniques Used: Expressing, Western Blot, Confocal Microscopy, Staining

    SUSD4 interacts with growth factor receptors. A Proximity ligation assay indicating interaction (white spots in maximum intensity projection of z-stack image) between SUSD4 and EGFR in both BT-20 and MDA-MB-468 cells stably expressing SUSD4 (every data point represents the average of at least 10 cells per sample). Sandwich ELISA using lysates of SUSD4-expressing BT-20 (B) or MDA-MB-468 cells (C) and corresponding mock control cells showing an interaction between SUSD4 and EGFR in both cell lines ( n = 3 biological repeats in each). D Proximity ligation assay indicative of an interaction (white spots in maximum intensity projection of z-stack image) between SUSD4 and PDGFRα in BT-20 cells and MDA-MB-468 cells stably expressing SUSD4 (Every data point represents the average of at least 10 cells per sample). E Sandwich ELISA showing an interaction between SUSD4 and PDGFRα in BT-20 cells expressing SUSD4. F Same ELISA as in (E) but including lysates of CRISPR/Cas9-mediated EGFR knockout cells stably expressing human SUSD4 and corresponding mock control cells. G Co-immunoprecipitation of the protein complexes between EGFR and SUSD4 using lysates of the BT-20 and MDA-MB-468 cells stably expressing SUSD4. All results were repeated in at least three independent biological experiments. The p -value was estimated with t test when two groups were compared and with Two-way ANOVA test when two or more groups were compared for two independent parameters * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001
    Figure Legend Snippet: SUSD4 interacts with growth factor receptors. A Proximity ligation assay indicating interaction (white spots in maximum intensity projection of z-stack image) between SUSD4 and EGFR in both BT-20 and MDA-MB-468 cells stably expressing SUSD4 (every data point represents the average of at least 10 cells per sample). Sandwich ELISA using lysates of SUSD4-expressing BT-20 (B) or MDA-MB-468 cells (C) and corresponding mock control cells showing an interaction between SUSD4 and EGFR in both cell lines ( n = 3 biological repeats in each). D Proximity ligation assay indicative of an interaction (white spots in maximum intensity projection of z-stack image) between SUSD4 and PDGFRα in BT-20 cells and MDA-MB-468 cells stably expressing SUSD4 (Every data point represents the average of at least 10 cells per sample). E Sandwich ELISA showing an interaction between SUSD4 and PDGFRα in BT-20 cells expressing SUSD4. F Same ELISA as in (E) but including lysates of CRISPR/Cas9-mediated EGFR knockout cells stably expressing human SUSD4 and corresponding mock control cells. G Co-immunoprecipitation of the protein complexes between EGFR and SUSD4 using lysates of the BT-20 and MDA-MB-468 cells stably expressing SUSD4. All results were repeated in at least three independent biological experiments. The p -value was estimated with t test when two groups were compared and with Two-way ANOVA test when two or more groups were compared for two independent parameters * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001

    Techniques Used: Proximity Ligation Assay, Stable Transfection, Expressing, Sandwich ELISA, Enzyme-linked Immunosorbent Assay, CRISPR, Knock-Out, Immunoprecipitation

    SUSD4’s effect on autophagy depends on its interaction with the EGFR. Autophagy in SUSD4-expressing BT-20 cells and mock control cells in addition to CRISPR/Cas9-mediated EGFR knockout BT-20 cells stably expressing SUSD4 and corresponding EGFR knockout mock control cells. Cells were treated with chloroquine for up to 5 hours and LC3B-II levels were assessed by immunoblot. Representative immunoblots (A) and densitometric analysis (B) of LC3B-II levels at each time-point. Total EGFR or a kinase-dead mutant EGFR was re-introduced in SUSD4-expressing BT-20 EGFR knockout cells and BT-20 EGFR knockout mock control cells. C Representative immunoblot verifying the presence of total-, or kinase-dead EGFR as well as showing LC3B-II levels in both untreated cells and cells treated with chloroquine for 4 hours. D Densitometric analysis of LC3B-II levels shown in (C) ( n = 4 biological repeats). Representative immunoblots (E) and densitometric analysis (F) of LC3B-II levels in untreated and chloroquine treated (4 hours) mock control and SUSD4-expressing BT-20 EGFR knockout cells transiently transfected with constructs allowing for the expression of myc-tagged intra- or extracellular domain of EGFR ( n = 4 biological repeats). The molecular weight of the EGFR ICD and ECD is 72 kDa and 80 kDa, respectively. G Autophagy in BT-20 cells expressing SUSD4 phosphorylation mutants SUSD4-LSPF (Y379F) or SUSD4-PPAF (Y414F). Immunoblot of SUSD4 phosphorylation mutants in addition to a representative immunoblot showing LC3B-II levels in untreated cells and cells treated with chloroquine for 3 hours. H Densitometric analysis of LC3B-II levels in BT-20 SUSD4 phosphorylation mutants shown in (G) . Deletion mutants of extracellular CCP domains of SUSD4 stably expressed in BT-20 cells. Autophagy was evaluated with LC3B western blots (I) in the presence or absence of chloroquine and band density relative to GAPDH was plotted (K) . All results were repeated in at least three independent biological experiments. The p -value was estimated with t test when two groups were compared and with Two-way ANOVA test when two or more groups were compared for two independent parameters * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001
    Figure Legend Snippet: SUSD4’s effect on autophagy depends on its interaction with the EGFR. Autophagy in SUSD4-expressing BT-20 cells and mock control cells in addition to CRISPR/Cas9-mediated EGFR knockout BT-20 cells stably expressing SUSD4 and corresponding EGFR knockout mock control cells. Cells were treated with chloroquine for up to 5 hours and LC3B-II levels were assessed by immunoblot. Representative immunoblots (A) and densitometric analysis (B) of LC3B-II levels at each time-point. Total EGFR or a kinase-dead mutant EGFR was re-introduced in SUSD4-expressing BT-20 EGFR knockout cells and BT-20 EGFR knockout mock control cells. C Representative immunoblot verifying the presence of total-, or kinase-dead EGFR as well as showing LC3B-II levels in both untreated cells and cells treated with chloroquine for 4 hours. D Densitometric analysis of LC3B-II levels shown in (C) ( n = 4 biological repeats). Representative immunoblots (E) and densitometric analysis (F) of LC3B-II levels in untreated and chloroquine treated (4 hours) mock control and SUSD4-expressing BT-20 EGFR knockout cells transiently transfected with constructs allowing for the expression of myc-tagged intra- or extracellular domain of EGFR ( n = 4 biological repeats). The molecular weight of the EGFR ICD and ECD is 72 kDa and 80 kDa, respectively. G Autophagy in BT-20 cells expressing SUSD4 phosphorylation mutants SUSD4-LSPF (Y379F) or SUSD4-PPAF (Y414F). Immunoblot of SUSD4 phosphorylation mutants in addition to a representative immunoblot showing LC3B-II levels in untreated cells and cells treated with chloroquine for 3 hours. H Densitometric analysis of LC3B-II levels in BT-20 SUSD4 phosphorylation mutants shown in (G) . Deletion mutants of extracellular CCP domains of SUSD4 stably expressed in BT-20 cells. Autophagy was evaluated with LC3B western blots (I) in the presence or absence of chloroquine and band density relative to GAPDH was plotted (K) . All results were repeated in at least three independent biological experiments. The p -value was estimated with t test when two groups were compared and with Two-way ANOVA test when two or more groups were compared for two independent parameters * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001

    Techniques Used: Expressing, CRISPR, Knock-Out, Stable Transfection, Western Blot, Mutagenesis, Transfection, Construct, Molecular Weight

    Phosphorylation status of signaling complexes in SUSD4-expressing cells favors autophagy initiation. A Comparison in phosphorylation status of a wide range of proteins in SUSD4-expressing BT-20 cells and mock control cells using the Human Phospho-Kinase Antibody Array ( n = 2 technical repeats). B-E Phospho-AMPKα1 levels in SUSD4-expressing BT-20 cells or MDA-MB-468 cells and corresponding mock control cells treated with AICAR (B and D) or A-769662 (C and E) . Representative immunoblots (B and C) and densitometric analysis (D and E) of phospho-AMPKα1 levels in both treated and untreated cells ( n = 3 biological repeats for treatment with A-769662 and BT-20 cells treated with AICAR, n = 4 biological repeats for MDA-MB-468 cells treated with AICAR). F and G Representative immunoblots (F) and densitometric analysis (G) showing the phosphorylation status of ULK1 at three distinct phosphorylation sites in BT-20 and MDA-MB-468 cells expressing SUSD4 and corresponding mock control cells ( n = 4 biological repeats for S757 and S555 in BT-20 cells, n = 3 biological repeats for S317 in BT-20 cells and all phosphorylation sites in MDA-MB-468 cells). H and I Representative immunoblot (H) and densitometric analysis (I) of phospho-activated Beclin-1 in BT-20 cells or MDA-MB-468 cells expressing SUSD4 compared to the respective mock control cells. J and K Representative immunoblot (J) and densitometric analysis (K) of phospho-Atg14 levels in BT-20 cells or MDA-MB-468 cells expressing SUSD4 and corresponding mock control cells. L-N Representative immunoblots and densitometric analyses showing phospho-LKB1 levels in BT-20 SUSD4 cells and mock control cells (L) , BT-20 EGFR knockout cells expressing or lacking SUSD4 (M) , and MDA-MB-468 cells expressing or lacking SUSD4. All results were repeated in at least three independent biological experiments. The p-value was estimated with t test when two groups were compared and with Two-way ANOVA test when two or more groups were compared for two independent parameters * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001
    Figure Legend Snippet: Phosphorylation status of signaling complexes in SUSD4-expressing cells favors autophagy initiation. A Comparison in phosphorylation status of a wide range of proteins in SUSD4-expressing BT-20 cells and mock control cells using the Human Phospho-Kinase Antibody Array ( n = 2 technical repeats). B-E Phospho-AMPKα1 levels in SUSD4-expressing BT-20 cells or MDA-MB-468 cells and corresponding mock control cells treated with AICAR (B and D) or A-769662 (C and E) . Representative immunoblots (B and C) and densitometric analysis (D and E) of phospho-AMPKα1 levels in both treated and untreated cells ( n = 3 biological repeats for treatment with A-769662 and BT-20 cells treated with AICAR, n = 4 biological repeats for MDA-MB-468 cells treated with AICAR). F and G Representative immunoblots (F) and densitometric analysis (G) showing the phosphorylation status of ULK1 at three distinct phosphorylation sites in BT-20 and MDA-MB-468 cells expressing SUSD4 and corresponding mock control cells ( n = 4 biological repeats for S757 and S555 in BT-20 cells, n = 3 biological repeats for S317 in BT-20 cells and all phosphorylation sites in MDA-MB-468 cells). H and I Representative immunoblot (H) and densitometric analysis (I) of phospho-activated Beclin-1 in BT-20 cells or MDA-MB-468 cells expressing SUSD4 compared to the respective mock control cells. J and K Representative immunoblot (J) and densitometric analysis (K) of phospho-Atg14 levels in BT-20 cells or MDA-MB-468 cells expressing SUSD4 and corresponding mock control cells. L-N Representative immunoblots and densitometric analyses showing phospho-LKB1 levels in BT-20 SUSD4 cells and mock control cells (L) , BT-20 EGFR knockout cells expressing or lacking SUSD4 (M) , and MDA-MB-468 cells expressing or lacking SUSD4. All results were repeated in at least three independent biological experiments. The p-value was estimated with t test when two groups were compared and with Two-way ANOVA test when two or more groups were compared for two independent parameters * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001

    Techniques Used: Expressing, Ab Array, Western Blot, Knock-Out

    SUSD4 colocalization with endo−/lysosomal markers. Representative merged confocal images showing EGFR (red), SUSD4 (green), endo−/lysosomal markers (blue) and DAPI (magenta) in BT-20 cells (A) or MDA-MB-468 cells (B) expressing SUSD4 and corresponding mock control cells. Note that the combination of the colors red, green, and blue is presented in white color. Colocalization coefficient for EGFR or SUSD4 with endo−/lysosomal markers quantified in BT-20 cells (C) and MDA-MB-468 cells (D) (Every data point represents the average of at least 10 cells per sample). All results were repeated in at least three independent biological experiments. Pearson’s correlation coefficient r where a minimal r value of 0.5 (dotted line) indicates significant colocalization
    Figure Legend Snippet: SUSD4 colocalization with endo−/lysosomal markers. Representative merged confocal images showing EGFR (red), SUSD4 (green), endo−/lysosomal markers (blue) and DAPI (magenta) in BT-20 cells (A) or MDA-MB-468 cells (B) expressing SUSD4 and corresponding mock control cells. Note that the combination of the colors red, green, and blue is presented in white color. Colocalization coefficient for EGFR or SUSD4 with endo−/lysosomal markers quantified in BT-20 cells (C) and MDA-MB-468 cells (D) (Every data point represents the average of at least 10 cells per sample). All results were repeated in at least three independent biological experiments. Pearson’s correlation coefficient r where a minimal r value of 0.5 (dotted line) indicates significant colocalization

    Techniques Used: Expressing

    bt 20  (ATCC)


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    ATCC bt 20
    Design of the study (A) MDA-MB-231, SUM149PT or <t>BT-20</t> 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 ( <xref ref-type=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. " width="250" height="auto" />
    Bt 20, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "AMPK is required for recovery from metabolic stress induced by ultrasound microbubble treatment"

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

    Journal: iScience

    doi: 10.1016/j.isci.2022.105883

    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 ( <xref ref-type=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. " title="Design of the study (A) MDA-MB-231, SUM149PT or BT-20 cells were prepared and treated with USMB using ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend 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.

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

    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.
    Figure Legend 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.

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

    bt 20  (ATCC)


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    ATCC bt 20
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    bt 20 cells  (ATCC)


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    human breast carcinoma cell lines  (ATCC)


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    ATCC human breast carcinoma cell lines
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    human breast cancer cell line bt  (ATCC)


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    ATCC human breast cancer cell line bt
    Effect of Tan IIA or STS on the growth of <t>BT-20</t> breast cancer cells. (a) BT-20 cells were treated with 2, 10, 25, and 50 μ g/mL of Tan IIA or STS for 72 h. The cell growth was determined by WST-1 assay. (b) BT-20 cells were treated with 2, 10, 25, and 50 μ g/mL of Tan IIA or STS for 24 h or 48 h. The ratio of apoptosis was determined by flow cytometry. Data are the mean ± SD of triplicates from a representative assay of three separate experiments.
    Human Breast Cancer Cell Line Bt, supplied by ATCC, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Effect of Supplementation of Tanshinone IIA and Sodium Tanshinone IIA Sulfonate on the Anticancer Effect of Epirubicin: An In Vitro Study"

    Article Title: Effect of Supplementation of Tanshinone IIA and Sodium Tanshinone IIA Sulfonate on the Anticancer Effect of Epirubicin: An In Vitro Study

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    doi: 10.1155/2011/841564

    Effect of Tan IIA or STS on the growth of BT-20 breast cancer cells. (a) BT-20 cells were treated with 2, 10, 25, and 50 μ g/mL of Tan IIA or STS for 72 h. The cell growth was determined by WST-1 assay. (b) BT-20 cells were treated with 2, 10, 25, and 50 μ g/mL of Tan IIA or STS for 24 h or 48 h. The ratio of apoptosis was determined by flow cytometry. Data are the mean ± SD of triplicates from a representative assay of three separate experiments.
    Figure Legend Snippet: Effect of Tan IIA or STS on the growth of BT-20 breast cancer cells. (a) BT-20 cells were treated with 2, 10, 25, and 50 μ g/mL of Tan IIA or STS for 72 h. The cell growth was determined by WST-1 assay. (b) BT-20 cells were treated with 2, 10, 25, and 50 μ g/mL of Tan IIA or STS for 24 h or 48 h. The ratio of apoptosis was determined by flow cytometry. Data are the mean ± SD of triplicates from a representative assay of three separate experiments.

    Techniques Used: WST-1 Assay, Flow Cytometry

    Effect of Tan IIA or STS on epirubicin-induced cytotoxicity and apoptosis in BT-20 breast cancer cells. (a) BT-20 breast cancer cells were treated with 0–2 μ g/mL epirubicin in the presence or absence of STS (0–50 μ g/mL) or Tan IIA (0–50 μ g/mL) for 72 h. The cell growth was determined by WST-1 assay. (b) BT-20 cells were treated with 1 μ g/mL of epirubicin. The change in apoptosis with the addition of STS (50 μ g/mL) and Tan IIA (2, 10, 25, 50 μ g/mL) in epirubicin- (Epi-) treated BT-20 cells was determined by flow cytometry at 48 or 72 h. * P < .05 when compared with the control group; + P < .05 when compared with the epirubicin group. Data are the mean ± SD of triplicates from a representative assay of three separate experiments.
    Figure Legend Snippet: Effect of Tan IIA or STS on epirubicin-induced cytotoxicity and apoptosis in BT-20 breast cancer cells. (a) BT-20 breast cancer cells were treated with 0–2 μ g/mL epirubicin in the presence or absence of STS (0–50 μ g/mL) or Tan IIA (0–50 μ g/mL) for 72 h. The cell growth was determined by WST-1 assay. (b) BT-20 cells were treated with 1 μ g/mL of epirubicin. The change in apoptosis with the addition of STS (50 μ g/mL) and Tan IIA (2, 10, 25, 50 μ g/mL) in epirubicin- (Epi-) treated BT-20 cells was determined by flow cytometry at 48 or 72 h. * P < .05 when compared with the control group; + P < .05 when compared with the epirubicin group. Data are the mean ± SD of triplicates from a representative assay of three separate experiments.

    Techniques Used: WST-1 Assay, Flow Cytometry

    Uptake of epirubicin in BT-20 breast cancer cells in the presence or absence of STS (a) or Tan IIA (b) treatment for 24 h. Data are the mean ± SD of triplicates from a representative assay of three separate experiments. * P < .05 as compared with 1 μ g/mL epirubicin-treated BT-20 cells.
    Figure Legend Snippet: Uptake of epirubicin in BT-20 breast cancer cells in the presence or absence of STS (a) or Tan IIA (b) treatment for 24 h. Data are the mean ± SD of triplicates from a representative assay of three separate experiments. * P < .05 as compared with 1 μ g/mL epirubicin-treated BT-20 cells.

    Techniques Used:

    Akt-related pathway activation in the presence or absence of STS (or Tan IIA) in epirubicin-treated BT-20 breast cancer cells for 24 h. (a) Representative results of Western blotting showing changes in the levels of Akt, phospho-Akt, JNK, phospho-JNK, P38, phospho-p38, MAPK, and phospho-MAPK in BT-20 cells treated with STS and/or 1 μ g/mL epirubicin. (b) Representative results of Western blotting showing changes in the levels of Akt, phospho-Akt, JNK, phospho-JNK, P38, phospho-p38, MAPK, and phospho-MAPK in BT-20 cells treated with Tan IIA and/or 1 μ g/mL epirubicin.
    Figure Legend Snippet: Akt-related pathway activation in the presence or absence of STS (or Tan IIA) in epirubicin-treated BT-20 breast cancer cells for 24 h. (a) Representative results of Western blotting showing changes in the levels of Akt, phospho-Akt, JNK, phospho-JNK, P38, phospho-p38, MAPK, and phospho-MAPK in BT-20 cells treated with STS and/or 1 μ g/mL epirubicin. (b) Representative results of Western blotting showing changes in the levels of Akt, phospho-Akt, JNK, phospho-JNK, P38, phospho-p38, MAPK, and phospho-MAPK in BT-20 cells treated with Tan IIA and/or 1 μ g/mL epirubicin.

    Techniques Used: Activation Assay, Western Blot

    bt 20  (ATCC)


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    bt 20  (ATCC)


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    86
    ATCC bt 474 cells cat htb 20 cells
    MCF-7 and <t>BT-474</t> cells were cultured on soft (0.5 kPa) or stiff (9 kPa) supports for 2 weeks before subsequent analysis. a Docetaxel (Doc) induced apoptosis of MCF-7 and BT-474 was assessed by flow cytometry. The proportions of Annexin V + cells are shown. b Quantitation of apoptotic cells (Annexin V + ) in MCF-7 and BT-474 cells in (a) ( n = 5 biologically independent experiments). Mean ± SD, **** p < 0.0001 by two-sided two-way ANOVA with Tukey test. c MCF-7 and BT-474 cells cultured on soft or stiff supports were treated with docetaxel or cisplatin. Cell viability was evaluated after 24 h by CCK8 assays ( n = 5 biologically independent experiments). Mean ± SD, **** p < 0.0001 by two-sided Student’s t test. d Western blots for cleaved/uncleaved caspase-3 and PARP in docetaxel-treated MCF-7 and BT-474 cells cultured on soft or stiff supports. Representative images from 3 biologically independent experiments are shown. e The percentage of CD44 high CD24 low cells in MCF-7 and BT-474 cells cultured on soft or stiff supports were determined by flow cytometry. f Quantitation of CD44 high CD24 low cells in (e) ( n = 5 biologically independent experiments). Mean ± SD, **** p < 0.0001 by two-sided Student’s t test. For b-d and f , source data are provided as a Source Data file.
    Bt 474 Cells Cat Htb 20 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    ATCC breast cancer cell lines bt 20
    SUSD4 expression suppresses tumor growth and affect EGFR dynamics. Tumor growth monitored in a syngeneic mouse model. 4 T1-Luc2 cells stably expressing mouse SUSD4, or mock control cells were injected into the mammary fat pad of BALB/c mice ( n = 10 mice per group). In first experiment ( A ) 2 × 10 6 cells were injected per mouse and in the second ( B ) 5 × 10 6 cells were injected per mouse (Error bars represents standard error of the mean). ( C ) Migration and invasion assay ( D ) of 5 × 10 4 4 T1-Luc2 cells incubated for 48 h under serum. (E) Immunodetection of SUSD4 from lysates of <t>BT-20</t> cells stably expressing human SUSD4 or mock control cells treated with deglycosylation enzymes to assess SUSD4 N-glycosylation status. (F) Protein profile comparison between SUSD4-expressing BT-20 cells and mock control cells using the Human XL Oncology Array ( n = 2 technical repeats). (G) Expression of SUSD4 and EGFR, independent of each other, in individual cell types present in breast cancer tumors. Representative immunoblots depicting total EGFR levels and phospho-EGFR at Tyr1045 (H) or Tyr1086 (I) in SUSD4-expressing BT-20 or MDA-MB-468 cells and corresponding mock control cells. Densitometric analysis of total EGFR and phospho-EGFR levels together with the ratio of phosphorylated EGFR to total EGFR in BT-20 cells (J) and MDA-MB-468 cells (K). (L) EGFR ubiquitination assessed by immunoprecipitation of EGFR in serum-starved EGF-treated BT-20 cell expressing SUSD4 or mock control cells followed by immunoblot detection of ubiquitin. All results were repeated in at least three independent biological experiments unless otherwise specified. The p -value was estimated with t test when two groups were compared and with Two-way ANOVA test when two or more groups were compared for two independent parameters * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001
    Breast Cancer Cell Lines Bt 20, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/breast cancer cell lines bt 20/product/ATCC
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    86
    ATCC bt 20 cells
    SUSD4 expression suppresses tumor growth and affect EGFR dynamics. Tumor growth monitored in a syngeneic mouse model. 4 T1-Luc2 cells stably expressing mouse SUSD4, or mock control cells were injected into the mammary fat pad of BALB/c mice ( n = 10 mice per group). In first experiment ( A ) 2 × 10 6 cells were injected per mouse and in the second ( B ) 5 × 10 6 cells were injected per mouse (Error bars represents standard error of the mean). ( C ) Migration and invasion assay ( D ) of 5 × 10 4 4 T1-Luc2 cells incubated for 48 h under serum. (E) Immunodetection of SUSD4 from lysates of <t>BT-20</t> cells stably expressing human SUSD4 or mock control cells treated with deglycosylation enzymes to assess SUSD4 N-glycosylation status. (F) Protein profile comparison between SUSD4-expressing BT-20 cells and mock control cells using the Human XL Oncology Array ( n = 2 technical repeats). (G) Expression of SUSD4 and EGFR, independent of each other, in individual cell types present in breast cancer tumors. Representative immunoblots depicting total EGFR levels and phospho-EGFR at Tyr1045 (H) or Tyr1086 (I) in SUSD4-expressing BT-20 or MDA-MB-468 cells and corresponding mock control cells. Densitometric analysis of total EGFR and phospho-EGFR levels together with the ratio of phosphorylated EGFR to total EGFR in BT-20 cells (J) and MDA-MB-468 cells (K). (L) EGFR ubiquitination assessed by immunoprecipitation of EGFR in serum-starved EGF-treated BT-20 cell expressing SUSD4 or mock control cells followed by immunoblot detection of ubiquitin. All results were repeated in at least three independent biological experiments unless otherwise specified. The p -value was estimated with t test when two groups were compared and with Two-way ANOVA test when two or more groups were compared for two independent parameters * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001
    Bt 20 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC human breast cancer cell line bt
    Effect of Tan IIA or STS on the growth of <t>BT-20</t> breast cancer cells. (a) BT-20 cells were treated with 2, 10, 25, and 50 μ g/mL of Tan IIA or STS for 72 h. The cell growth was determined by WST-1 assay. (b) BT-20 cells were treated with 2, 10, 25, and 50 μ g/mL of Tan IIA or STS for 24 h or 48 h. The ratio of apoptosis was determined by flow cytometry. Data are the mean ± SD of triplicates from a representative assay of three separate experiments.
    Human Breast Cancer Cell Line Bt, supplied by ATCC, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MCF-7 and BT-474 cells were cultured on soft (0.5 kPa) or stiff (9 kPa) supports for 2 weeks before subsequent analysis. a Docetaxel (Doc) induced apoptosis of MCF-7 and BT-474 was assessed by flow cytometry. The proportions of Annexin V + cells are shown. b Quantitation of apoptotic cells (Annexin V + ) in MCF-7 and BT-474 cells in (a) ( n = 5 biologically independent experiments). Mean ± SD, **** p < 0.0001 by two-sided two-way ANOVA with Tukey test. c MCF-7 and BT-474 cells cultured on soft or stiff supports were treated with docetaxel or cisplatin. Cell viability was evaluated after 24 h by CCK8 assays ( n = 5 biologically independent experiments). Mean ± SD, **** p < 0.0001 by two-sided Student’s t test. d Western blots for cleaved/uncleaved caspase-3 and PARP in docetaxel-treated MCF-7 and BT-474 cells cultured on soft or stiff supports. Representative images from 3 biologically independent experiments are shown. e The percentage of CD44 high CD24 low cells in MCF-7 and BT-474 cells cultured on soft or stiff supports were determined by flow cytometry. f Quantitation of CD44 high CD24 low cells in (e) ( n = 5 biologically independent experiments). Mean ± SD, **** p < 0.0001 by two-sided Student’s t test. For b-d and f , source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: Niche stiffness sustains cancer stemness via TAZ and NANOG phase separation

    doi: 10.1038/s41467-023-35856-y

    Figure Lengend Snippet: MCF-7 and BT-474 cells were cultured on soft (0.5 kPa) or stiff (9 kPa) supports for 2 weeks before subsequent analysis. a Docetaxel (Doc) induced apoptosis of MCF-7 and BT-474 was assessed by flow cytometry. The proportions of Annexin V + cells are shown. b Quantitation of apoptotic cells (Annexin V + ) in MCF-7 and BT-474 cells in (a) ( n = 5 biologically independent experiments). Mean ± SD, **** p < 0.0001 by two-sided two-way ANOVA with Tukey test. c MCF-7 and BT-474 cells cultured on soft or stiff supports were treated with docetaxel or cisplatin. Cell viability was evaluated after 24 h by CCK8 assays ( n = 5 biologically independent experiments). Mean ± SD, **** p < 0.0001 by two-sided Student’s t test. d Western blots for cleaved/uncleaved caspase-3 and PARP in docetaxel-treated MCF-7 and BT-474 cells cultured on soft or stiff supports. Representative images from 3 biologically independent experiments are shown. e The percentage of CD44 high CD24 low cells in MCF-7 and BT-474 cells cultured on soft or stiff supports were determined by flow cytometry. f Quantitation of CD44 high CD24 low cells in (e) ( n = 5 biologically independent experiments). Mean ± SD, **** p < 0.0001 by two-sided Student’s t test. For b-d and f , source data are provided as a Source Data file.

    Article Snippet: MCF-7 cells (cat#HTB-22) and BT-474 cells (cat#HTB-20) cells were obtained from American Type Culture Collection (ATCC) and cultured according to standard protocols.

    Techniques: Cell Culture, Flow Cytometry, Quantitation Assay, Western Blot

    MCF-7 and BT-474 cells were cultured on soft or stiff supports for 2 weeks before subsequent analysis. In some experiments, cells were pre-transduced with TAZ shRNA (shTAZ-1 or shTAZ-2) or GFP shRNA (shGFP). a The expression of TAZ in MCF-7 and BT-474 cells cultured on soft or stiff supports was determined by western blot ( n = 3 biologically independent experiments). b Representative immunofluorescence staining of TAZ in MCF-7 and BT-474 cells cultured on soft or stiff substrates. The insert represents a magnification from the indicated area (white square). Scale bars, 10 μm. c Proportion of cancer cells exhibiting mainly nuclear TAZ localization (N > C), diffuse distribution of TAZ in nucleus and cytoplasm (N = C), or preferential cytoplasmic TAZ (N < C or undetectable). 20 cells were assessed per group, experiments were independently repeated 4 times. Mean ± SD, **** p < 0.0001 by two-tailed chi-square test. d Docetaxel induced apoptosis of MCF-7 and BT-474 was assessed by flow cytometry. See Supplementary Fig. for quantification. e MCF-7 and BT-474 cells cultured on soft or stiff supports were treated with docetaxel or cisplatin. Cell viability was evaluated by CCK8 assay ( n = 5 biologically independent experiments). Mean ± SD, **** p < 0.0001 by two-sided one-way ANOVA with Tukey test. f Representative images of CD44 high CD24 low cells in MCF-7 and BT-474 cells were detected by flow cytometry. See Supplementary Fig. for quantification. g Western blots of TAZ, SOX2, and OCT4 in MCF-7 and BT-474 cells cultured on soft or stiff supports ( n = 3 biologically independent experiments). For data presented in the same figure panel, the samples were derived from the same experiment and blots were processed in parallel. For a , c , e and g , source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: Niche stiffness sustains cancer stemness via TAZ and NANOG phase separation

    doi: 10.1038/s41467-023-35856-y

    Figure Lengend Snippet: MCF-7 and BT-474 cells were cultured on soft or stiff supports for 2 weeks before subsequent analysis. In some experiments, cells were pre-transduced with TAZ shRNA (shTAZ-1 or shTAZ-2) or GFP shRNA (shGFP). a The expression of TAZ in MCF-7 and BT-474 cells cultured on soft or stiff supports was determined by western blot ( n = 3 biologically independent experiments). b Representative immunofluorescence staining of TAZ in MCF-7 and BT-474 cells cultured on soft or stiff substrates. The insert represents a magnification from the indicated area (white square). Scale bars, 10 μm. c Proportion of cancer cells exhibiting mainly nuclear TAZ localization (N > C), diffuse distribution of TAZ in nucleus and cytoplasm (N = C), or preferential cytoplasmic TAZ (N < C or undetectable). 20 cells were assessed per group, experiments were independently repeated 4 times. Mean ± SD, **** p < 0.0001 by two-tailed chi-square test. d Docetaxel induced apoptosis of MCF-7 and BT-474 was assessed by flow cytometry. See Supplementary Fig. for quantification. e MCF-7 and BT-474 cells cultured on soft or stiff supports were treated with docetaxel or cisplatin. Cell viability was evaluated by CCK8 assay ( n = 5 biologically independent experiments). Mean ± SD, **** p < 0.0001 by two-sided one-way ANOVA with Tukey test. f Representative images of CD44 high CD24 low cells in MCF-7 and BT-474 cells were detected by flow cytometry. See Supplementary Fig. for quantification. g Western blots of TAZ, SOX2, and OCT4 in MCF-7 and BT-474 cells cultured on soft or stiff supports ( n = 3 biologically independent experiments). For data presented in the same figure panel, the samples were derived from the same experiment and blots were processed in parallel. For a , c , e and g , source data are provided as a Source Data file.

    Article Snippet: MCF-7 cells (cat#HTB-22) and BT-474 cells (cat#HTB-20) cells were obtained from American Type Culture Collection (ATCC) and cultured according to standard protocols.

    Techniques: Cell Culture, Transduction, shRNA, Expressing, Western Blot, Immunofluorescence, Staining, Two Tailed Test, Flow Cytometry, CCK-8 Assay, Derivative Assay

    a – d TAZ and NANOG were knockout or overexpressed by lentiviral transfection in MCF-7 and BT-474 cells. NANOG, TAZ, SOX2 or/and OCT4 were overexpressed in TAZ or NANOG knockout cells. Transfected cells were cultured on stiff hydrogels for 2 weeks. OE, overexpression. a (Left) Representative flow cytometric plot indicating the percentages of CD44 high CD24 low cells in MCF-7 and BT-474 cells. (Right) Qualification of five biologically independent experiments. **** p < 0.0001; for MCF-7 cells, *** p = 0.0003, ** p = 0.0039, ns=0.9899; for BT-474 cells, *** p = 0.0008, ns=0.9982. b Cell viability of MCF-7 and BT-474 cells treated with various concentrations of docetaxel and cisplatin for 24 h was determined by CCK8 assays ( n = 4 biologically independent experiments). P values compared with sgTAZ + Vector group were shown in the figure. c (Left) Representative flow cytometric plot indicating the percentages of CD44 high CD24 low cells in MCF-7 and BT-474 cells. (Right) Qualification of five independent experiments. **** p < 0.0001; for MCF-7 cells, *** p = 0.0004, ** p = 0.0033, ns=0.9400; for BT-474 cells, *** p = 0.0002, ** p = 0.0013, ns=0.9982. d Cell viability of MCF-7 and BT-474 cells treated with various concentrations of docetaxel and cisplatin for 24 h was determined by CCK8 assays ( n = 4 biologically independent experiments). P values compared with sgNANOG + Vector group were shown in the figure. For panels (a– d) , data are mean ± SD; p values were calculated by two-sided one-way ANOVA with Tukey test. e , f MCF-7 cells were cultured on soft or stiff hydrogels for 2 weeks. Then ChIP was performed using cell lysates with indicated antibodies. e The precipitated DNA using IgG and TAZ antibody, respectively, was quantified by qPCR with primers specific to promoter regions of Sox2 and Pou5f1 . f The precipitated DNA using IgG and NANOG antibody, respectively, was quantified by qPCR with primers specific to promoter regions of Sox2 and Pou5f1 . e , f Fold enrichment was normalized to IgG-ChIP negative control ( n = 4 biologically independent experiments). Numeric values denote mean ± SD; **** p < 0.0001 by two-sided two-way ANOVA with Tukey test. For a–f , source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: Niche stiffness sustains cancer stemness via TAZ and NANOG phase separation

    doi: 10.1038/s41467-023-35856-y

    Figure Lengend Snippet: a – d TAZ and NANOG were knockout or overexpressed by lentiviral transfection in MCF-7 and BT-474 cells. NANOG, TAZ, SOX2 or/and OCT4 were overexpressed in TAZ or NANOG knockout cells. Transfected cells were cultured on stiff hydrogels for 2 weeks. OE, overexpression. a (Left) Representative flow cytometric plot indicating the percentages of CD44 high CD24 low cells in MCF-7 and BT-474 cells. (Right) Qualification of five biologically independent experiments. **** p < 0.0001; for MCF-7 cells, *** p = 0.0003, ** p = 0.0039, ns=0.9899; for BT-474 cells, *** p = 0.0008, ns=0.9982. b Cell viability of MCF-7 and BT-474 cells treated with various concentrations of docetaxel and cisplatin for 24 h was determined by CCK8 assays ( n = 4 biologically independent experiments). P values compared with sgTAZ + Vector group were shown in the figure. c (Left) Representative flow cytometric plot indicating the percentages of CD44 high CD24 low cells in MCF-7 and BT-474 cells. (Right) Qualification of five independent experiments. **** p < 0.0001; for MCF-7 cells, *** p = 0.0004, ** p = 0.0033, ns=0.9400; for BT-474 cells, *** p = 0.0002, ** p = 0.0013, ns=0.9982. d Cell viability of MCF-7 and BT-474 cells treated with various concentrations of docetaxel and cisplatin for 24 h was determined by CCK8 assays ( n = 4 biologically independent experiments). P values compared with sgNANOG + Vector group were shown in the figure. For panels (a– d) , data are mean ± SD; p values were calculated by two-sided one-way ANOVA with Tukey test. e , f MCF-7 cells were cultured on soft or stiff hydrogels for 2 weeks. Then ChIP was performed using cell lysates with indicated antibodies. e The precipitated DNA using IgG and TAZ antibody, respectively, was quantified by qPCR with primers specific to promoter regions of Sox2 and Pou5f1 . f The precipitated DNA using IgG and NANOG antibody, respectively, was quantified by qPCR with primers specific to promoter regions of Sox2 and Pou5f1 . e , f Fold enrichment was normalized to IgG-ChIP negative control ( n = 4 biologically independent experiments). Numeric values denote mean ± SD; **** p < 0.0001 by two-sided two-way ANOVA with Tukey test. For a–f , source data are provided as a Source Data file.

    Article Snippet: MCF-7 cells (cat#HTB-22) and BT-474 cells (cat#HTB-20) cells were obtained from American Type Culture Collection (ATCC) and cultured according to standard protocols.

    Techniques: Knock-Out, Transfection, Cell Culture, Over Expression, Plasmid Preparation, Negative Control

    SUSD4 expression suppresses tumor growth and affect EGFR dynamics. Tumor growth monitored in a syngeneic mouse model. 4 T1-Luc2 cells stably expressing mouse SUSD4, or mock control cells were injected into the mammary fat pad of BALB/c mice ( n = 10 mice per group). In first experiment ( A ) 2 × 10 6 cells were injected per mouse and in the second ( B ) 5 × 10 6 cells were injected per mouse (Error bars represents standard error of the mean). ( C ) Migration and invasion assay ( D ) of 5 × 10 4 4 T1-Luc2 cells incubated for 48 h under serum. (E) Immunodetection of SUSD4 from lysates of BT-20 cells stably expressing human SUSD4 or mock control cells treated with deglycosylation enzymes to assess SUSD4 N-glycosylation status. (F) Protein profile comparison between SUSD4-expressing BT-20 cells and mock control cells using the Human XL Oncology Array ( n = 2 technical repeats). (G) Expression of SUSD4 and EGFR, independent of each other, in individual cell types present in breast cancer tumors. Representative immunoblots depicting total EGFR levels and phospho-EGFR at Tyr1045 (H) or Tyr1086 (I) in SUSD4-expressing BT-20 or MDA-MB-468 cells and corresponding mock control cells. Densitometric analysis of total EGFR and phospho-EGFR levels together with the ratio of phosphorylated EGFR to total EGFR in BT-20 cells (J) and MDA-MB-468 cells (K). (L) EGFR ubiquitination assessed by immunoprecipitation of EGFR in serum-starved EGF-treated BT-20 cell expressing SUSD4 or mock control cells followed by immunoblot detection of ubiquitin. All results were repeated in at least three independent biological experiments unless otherwise specified. The p -value was estimated with t test when two groups were compared and with Two-way ANOVA test when two or more groups were compared for two independent parameters * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Sushi domain-containing protein 4 binds to epithelial growth factor receptor and initiates autophagy in an EGFR phosphorylation independent manner

    doi: 10.1186/s13046-022-02565-1

    Figure Lengend Snippet: SUSD4 expression suppresses tumor growth and affect EGFR dynamics. Tumor growth monitored in a syngeneic mouse model. 4 T1-Luc2 cells stably expressing mouse SUSD4, or mock control cells were injected into the mammary fat pad of BALB/c mice ( n = 10 mice per group). In first experiment ( A ) 2 × 10 6 cells were injected per mouse and in the second ( B ) 5 × 10 6 cells were injected per mouse (Error bars represents standard error of the mean). ( C ) Migration and invasion assay ( D ) of 5 × 10 4 4 T1-Luc2 cells incubated for 48 h under serum. (E) Immunodetection of SUSD4 from lysates of BT-20 cells stably expressing human SUSD4 or mock control cells treated with deglycosylation enzymes to assess SUSD4 N-glycosylation status. (F) Protein profile comparison between SUSD4-expressing BT-20 cells and mock control cells using the Human XL Oncology Array ( n = 2 technical repeats). (G) Expression of SUSD4 and EGFR, independent of each other, in individual cell types present in breast cancer tumors. Representative immunoblots depicting total EGFR levels and phospho-EGFR at Tyr1045 (H) or Tyr1086 (I) in SUSD4-expressing BT-20 or MDA-MB-468 cells and corresponding mock control cells. Densitometric analysis of total EGFR and phospho-EGFR levels together with the ratio of phosphorylated EGFR to total EGFR in BT-20 cells (J) and MDA-MB-468 cells (K). (L) EGFR ubiquitination assessed by immunoprecipitation of EGFR in serum-starved EGF-treated BT-20 cell expressing SUSD4 or mock control cells followed by immunoblot detection of ubiquitin. All results were repeated in at least three independent biological experiments unless otherwise specified. The p -value was estimated with t test when two groups were compared and with Two-way ANOVA test when two or more groups were compared for two independent parameters * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001

    Article Snippet: Breast cancer cell lines BT-20 (ATCC), HS-578 T and MDA-MB-468 (DSMZ) were obtained directly from vendors and maintained in culture for a limited number of passages.

    Techniques: Expressing, Stable Transfection, Injection, Migration, Invasion Assay, Incubation, Immunodetection, Western Blot, Immunoprecipitation

    SUSD4 expression leads to increased autophagic flux. Autophagy levels SUSD4-expressing BT-20 cells (A and C) or MDA-MB-468 cells (B and D) and corresponding mock control cells treated with chloroquine for up to 5 hours. Representative immunoblots (A and B) and densitometric analysis (C and D) of LC3B-II levels in SUSD4-expressing cells and mock control cells for each cell line and chloroquine treatment time. Autophagy levels depicted by p62 levels in SUSD4-expressing BT-20 cells (E and G) or MDA-MB-468 cells (F and H) and corresponding mock control cells treated with chloroquine for 2- or 4 hours. Representative immunoblots and densitometric analysis of p62 levels in BT-20 cells (E and G) and MDA-MB-468 cells (F and H) . LC3B puncta in SUSD4-expressing BT-20 cells or MDA-MB-468 cells and corresponding mock control cells expressing GPF-tagged LC3B visualized by confocal microscopy. Representative images showing LC3B puncta (green) and DAPI (blue) (I) as well as quantified number of puncta per cell (J) in untreated cells and cells treated with chloroquine for 3 hours (Every data point represents the average of puncta per cell from at least 10 cells per sample). Assessment of autophagosome-lysosome fusion in BT-20 cells or MDA-MB-468 cell expressing SUSD4 and corresponding mock control cells using the Premo Autophagy Tandem Sensor RFP-GFP-LC3B (K), created with BioRender.com . Representative images (L) and quantified colocalization coefficient (M) of magenta- and green-stained vesicles. (Every data point represents the average of at least 10 cells per sample). All results were repeated in at least three independent biological experiments. The p-value was estimated with t test when two groups were compared and with Two-way ANOVA test when two or more groups were compared for two independent parameters * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Sushi domain-containing protein 4 binds to epithelial growth factor receptor and initiates autophagy in an EGFR phosphorylation independent manner

    doi: 10.1186/s13046-022-02565-1

    Figure Lengend Snippet: SUSD4 expression leads to increased autophagic flux. Autophagy levels SUSD4-expressing BT-20 cells (A and C) or MDA-MB-468 cells (B and D) and corresponding mock control cells treated with chloroquine for up to 5 hours. Representative immunoblots (A and B) and densitometric analysis (C and D) of LC3B-II levels in SUSD4-expressing cells and mock control cells for each cell line and chloroquine treatment time. Autophagy levels depicted by p62 levels in SUSD4-expressing BT-20 cells (E and G) or MDA-MB-468 cells (F and H) and corresponding mock control cells treated with chloroquine for 2- or 4 hours. Representative immunoblots and densitometric analysis of p62 levels in BT-20 cells (E and G) and MDA-MB-468 cells (F and H) . LC3B puncta in SUSD4-expressing BT-20 cells or MDA-MB-468 cells and corresponding mock control cells expressing GPF-tagged LC3B visualized by confocal microscopy. Representative images showing LC3B puncta (green) and DAPI (blue) (I) as well as quantified number of puncta per cell (J) in untreated cells and cells treated with chloroquine for 3 hours (Every data point represents the average of puncta per cell from at least 10 cells per sample). Assessment of autophagosome-lysosome fusion in BT-20 cells or MDA-MB-468 cell expressing SUSD4 and corresponding mock control cells using the Premo Autophagy Tandem Sensor RFP-GFP-LC3B (K), created with BioRender.com . Representative images (L) and quantified colocalization coefficient (M) of magenta- and green-stained vesicles. (Every data point represents the average of at least 10 cells per sample). All results were repeated in at least three independent biological experiments. The p-value was estimated with t test when two groups were compared and with Two-way ANOVA test when two or more groups were compared for two independent parameters * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001

    Article Snippet: Breast cancer cell lines BT-20 (ATCC), HS-578 T and MDA-MB-468 (DSMZ) were obtained directly from vendors and maintained in culture for a limited number of passages.

    Techniques: Expressing, Western Blot, Confocal Microscopy, Staining

    SUSD4 interacts with growth factor receptors. A Proximity ligation assay indicating interaction (white spots in maximum intensity projection of z-stack image) between SUSD4 and EGFR in both BT-20 and MDA-MB-468 cells stably expressing SUSD4 (every data point represents the average of at least 10 cells per sample). Sandwich ELISA using lysates of SUSD4-expressing BT-20 (B) or MDA-MB-468 cells (C) and corresponding mock control cells showing an interaction between SUSD4 and EGFR in both cell lines ( n = 3 biological repeats in each). D Proximity ligation assay indicative of an interaction (white spots in maximum intensity projection of z-stack image) between SUSD4 and PDGFRα in BT-20 cells and MDA-MB-468 cells stably expressing SUSD4 (Every data point represents the average of at least 10 cells per sample). E Sandwich ELISA showing an interaction between SUSD4 and PDGFRα in BT-20 cells expressing SUSD4. F Same ELISA as in (E) but including lysates of CRISPR/Cas9-mediated EGFR knockout cells stably expressing human SUSD4 and corresponding mock control cells. G Co-immunoprecipitation of the protein complexes between EGFR and SUSD4 using lysates of the BT-20 and MDA-MB-468 cells stably expressing SUSD4. All results were repeated in at least three independent biological experiments. The p -value was estimated with t test when two groups were compared and with Two-way ANOVA test when two or more groups were compared for two independent parameters * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Sushi domain-containing protein 4 binds to epithelial growth factor receptor and initiates autophagy in an EGFR phosphorylation independent manner

    doi: 10.1186/s13046-022-02565-1

    Figure Lengend Snippet: SUSD4 interacts with growth factor receptors. A Proximity ligation assay indicating interaction (white spots in maximum intensity projection of z-stack image) between SUSD4 and EGFR in both BT-20 and MDA-MB-468 cells stably expressing SUSD4 (every data point represents the average of at least 10 cells per sample). Sandwich ELISA using lysates of SUSD4-expressing BT-20 (B) or MDA-MB-468 cells (C) and corresponding mock control cells showing an interaction between SUSD4 and EGFR in both cell lines ( n = 3 biological repeats in each). D Proximity ligation assay indicative of an interaction (white spots in maximum intensity projection of z-stack image) between SUSD4 and PDGFRα in BT-20 cells and MDA-MB-468 cells stably expressing SUSD4 (Every data point represents the average of at least 10 cells per sample). E Sandwich ELISA showing an interaction between SUSD4 and PDGFRα in BT-20 cells expressing SUSD4. F Same ELISA as in (E) but including lysates of CRISPR/Cas9-mediated EGFR knockout cells stably expressing human SUSD4 and corresponding mock control cells. G Co-immunoprecipitation of the protein complexes between EGFR and SUSD4 using lysates of the BT-20 and MDA-MB-468 cells stably expressing SUSD4. All results were repeated in at least three independent biological experiments. The p -value was estimated with t test when two groups were compared and with Two-way ANOVA test when two or more groups were compared for two independent parameters * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001

    Article Snippet: Breast cancer cell lines BT-20 (ATCC), HS-578 T and MDA-MB-468 (DSMZ) were obtained directly from vendors and maintained in culture for a limited number of passages.

    Techniques: Proximity Ligation Assay, Stable Transfection, Expressing, Sandwich ELISA, Enzyme-linked Immunosorbent Assay, CRISPR, Knock-Out, Immunoprecipitation

    SUSD4’s effect on autophagy depends on its interaction with the EGFR. Autophagy in SUSD4-expressing BT-20 cells and mock control cells in addition to CRISPR/Cas9-mediated EGFR knockout BT-20 cells stably expressing SUSD4 and corresponding EGFR knockout mock control cells. Cells were treated with chloroquine for up to 5 hours and LC3B-II levels were assessed by immunoblot. Representative immunoblots (A) and densitometric analysis (B) of LC3B-II levels at each time-point. Total EGFR or a kinase-dead mutant EGFR was re-introduced in SUSD4-expressing BT-20 EGFR knockout cells and BT-20 EGFR knockout mock control cells. C Representative immunoblot verifying the presence of total-, or kinase-dead EGFR as well as showing LC3B-II levels in both untreated cells and cells treated with chloroquine for 4 hours. D Densitometric analysis of LC3B-II levels shown in (C) ( n = 4 biological repeats). Representative immunoblots (E) and densitometric analysis (F) of LC3B-II levels in untreated and chloroquine treated (4 hours) mock control and SUSD4-expressing BT-20 EGFR knockout cells transiently transfected with constructs allowing for the expression of myc-tagged intra- or extracellular domain of EGFR ( n = 4 biological repeats). The molecular weight of the EGFR ICD and ECD is 72 kDa and 80 kDa, respectively. G Autophagy in BT-20 cells expressing SUSD4 phosphorylation mutants SUSD4-LSPF (Y379F) or SUSD4-PPAF (Y414F). Immunoblot of SUSD4 phosphorylation mutants in addition to a representative immunoblot showing LC3B-II levels in untreated cells and cells treated with chloroquine for 3 hours. H Densitometric analysis of LC3B-II levels in BT-20 SUSD4 phosphorylation mutants shown in (G) . Deletion mutants of extracellular CCP domains of SUSD4 stably expressed in BT-20 cells. Autophagy was evaluated with LC3B western blots (I) in the presence or absence of chloroquine and band density relative to GAPDH was plotted (K) . All results were repeated in at least three independent biological experiments. The p -value was estimated with t test when two groups were compared and with Two-way ANOVA test when two or more groups were compared for two independent parameters * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Sushi domain-containing protein 4 binds to epithelial growth factor receptor and initiates autophagy in an EGFR phosphorylation independent manner

    doi: 10.1186/s13046-022-02565-1

    Figure Lengend Snippet: SUSD4’s effect on autophagy depends on its interaction with the EGFR. Autophagy in SUSD4-expressing BT-20 cells and mock control cells in addition to CRISPR/Cas9-mediated EGFR knockout BT-20 cells stably expressing SUSD4 and corresponding EGFR knockout mock control cells. Cells were treated with chloroquine for up to 5 hours and LC3B-II levels were assessed by immunoblot. Representative immunoblots (A) and densitometric analysis (B) of LC3B-II levels at each time-point. Total EGFR or a kinase-dead mutant EGFR was re-introduced in SUSD4-expressing BT-20 EGFR knockout cells and BT-20 EGFR knockout mock control cells. C Representative immunoblot verifying the presence of total-, or kinase-dead EGFR as well as showing LC3B-II levels in both untreated cells and cells treated with chloroquine for 4 hours. D Densitometric analysis of LC3B-II levels shown in (C) ( n = 4 biological repeats). Representative immunoblots (E) and densitometric analysis (F) of LC3B-II levels in untreated and chloroquine treated (4 hours) mock control and SUSD4-expressing BT-20 EGFR knockout cells transiently transfected with constructs allowing for the expression of myc-tagged intra- or extracellular domain of EGFR ( n = 4 biological repeats). The molecular weight of the EGFR ICD and ECD is 72 kDa and 80 kDa, respectively. G Autophagy in BT-20 cells expressing SUSD4 phosphorylation mutants SUSD4-LSPF (Y379F) or SUSD4-PPAF (Y414F). Immunoblot of SUSD4 phosphorylation mutants in addition to a representative immunoblot showing LC3B-II levels in untreated cells and cells treated with chloroquine for 3 hours. H Densitometric analysis of LC3B-II levels in BT-20 SUSD4 phosphorylation mutants shown in (G) . Deletion mutants of extracellular CCP domains of SUSD4 stably expressed in BT-20 cells. Autophagy was evaluated with LC3B western blots (I) in the presence or absence of chloroquine and band density relative to GAPDH was plotted (K) . All results were repeated in at least three independent biological experiments. The p -value was estimated with t test when two groups were compared and with Two-way ANOVA test when two or more groups were compared for two independent parameters * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001

    Article Snippet: Breast cancer cell lines BT-20 (ATCC), HS-578 T and MDA-MB-468 (DSMZ) were obtained directly from vendors and maintained in culture for a limited number of passages.

    Techniques: Expressing, CRISPR, Knock-Out, Stable Transfection, Western Blot, Mutagenesis, Transfection, Construct, Molecular Weight

    Phosphorylation status of signaling complexes in SUSD4-expressing cells favors autophagy initiation. A Comparison in phosphorylation status of a wide range of proteins in SUSD4-expressing BT-20 cells and mock control cells using the Human Phospho-Kinase Antibody Array ( n = 2 technical repeats). B-E Phospho-AMPKα1 levels in SUSD4-expressing BT-20 cells or MDA-MB-468 cells and corresponding mock control cells treated with AICAR (B and D) or A-769662 (C and E) . Representative immunoblots (B and C) and densitometric analysis (D and E) of phospho-AMPKα1 levels in both treated and untreated cells ( n = 3 biological repeats for treatment with A-769662 and BT-20 cells treated with AICAR, n = 4 biological repeats for MDA-MB-468 cells treated with AICAR). F and G Representative immunoblots (F) and densitometric analysis (G) showing the phosphorylation status of ULK1 at three distinct phosphorylation sites in BT-20 and MDA-MB-468 cells expressing SUSD4 and corresponding mock control cells ( n = 4 biological repeats for S757 and S555 in BT-20 cells, n = 3 biological repeats for S317 in BT-20 cells and all phosphorylation sites in MDA-MB-468 cells). H and I Representative immunoblot (H) and densitometric analysis (I) of phospho-activated Beclin-1 in BT-20 cells or MDA-MB-468 cells expressing SUSD4 compared to the respective mock control cells. J and K Representative immunoblot (J) and densitometric analysis (K) of phospho-Atg14 levels in BT-20 cells or MDA-MB-468 cells expressing SUSD4 and corresponding mock control cells. L-N Representative immunoblots and densitometric analyses showing phospho-LKB1 levels in BT-20 SUSD4 cells and mock control cells (L) , BT-20 EGFR knockout cells expressing or lacking SUSD4 (M) , and MDA-MB-468 cells expressing or lacking SUSD4. All results were repeated in at least three independent biological experiments. The p-value was estimated with t test when two groups were compared and with Two-way ANOVA test when two or more groups were compared for two independent parameters * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Sushi domain-containing protein 4 binds to epithelial growth factor receptor and initiates autophagy in an EGFR phosphorylation independent manner

    doi: 10.1186/s13046-022-02565-1

    Figure Lengend Snippet: Phosphorylation status of signaling complexes in SUSD4-expressing cells favors autophagy initiation. A Comparison in phosphorylation status of a wide range of proteins in SUSD4-expressing BT-20 cells and mock control cells using the Human Phospho-Kinase Antibody Array ( n = 2 technical repeats). B-E Phospho-AMPKα1 levels in SUSD4-expressing BT-20 cells or MDA-MB-468 cells and corresponding mock control cells treated with AICAR (B and D) or A-769662 (C and E) . Representative immunoblots (B and C) and densitometric analysis (D and E) of phospho-AMPKα1 levels in both treated and untreated cells ( n = 3 biological repeats for treatment with A-769662 and BT-20 cells treated with AICAR, n = 4 biological repeats for MDA-MB-468 cells treated with AICAR). F and G Representative immunoblots (F) and densitometric analysis (G) showing the phosphorylation status of ULK1 at three distinct phosphorylation sites in BT-20 and MDA-MB-468 cells expressing SUSD4 and corresponding mock control cells ( n = 4 biological repeats for S757 and S555 in BT-20 cells, n = 3 biological repeats for S317 in BT-20 cells and all phosphorylation sites in MDA-MB-468 cells). H and I Representative immunoblot (H) and densitometric analysis (I) of phospho-activated Beclin-1 in BT-20 cells or MDA-MB-468 cells expressing SUSD4 compared to the respective mock control cells. J and K Representative immunoblot (J) and densitometric analysis (K) of phospho-Atg14 levels in BT-20 cells or MDA-MB-468 cells expressing SUSD4 and corresponding mock control cells. L-N Representative immunoblots and densitometric analyses showing phospho-LKB1 levels in BT-20 SUSD4 cells and mock control cells (L) , BT-20 EGFR knockout cells expressing or lacking SUSD4 (M) , and MDA-MB-468 cells expressing or lacking SUSD4. All results were repeated in at least three independent biological experiments. The p-value was estimated with t test when two groups were compared and with Two-way ANOVA test when two or more groups were compared for two independent parameters * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001

    Article Snippet: Breast cancer cell lines BT-20 (ATCC), HS-578 T and MDA-MB-468 (DSMZ) were obtained directly from vendors and maintained in culture for a limited number of passages.

    Techniques: Expressing, Ab Array, Western Blot, Knock-Out

    SUSD4 colocalization with endo−/lysosomal markers. Representative merged confocal images showing EGFR (red), SUSD4 (green), endo−/lysosomal markers (blue) and DAPI (magenta) in BT-20 cells (A) or MDA-MB-468 cells (B) expressing SUSD4 and corresponding mock control cells. Note that the combination of the colors red, green, and blue is presented in white color. Colocalization coefficient for EGFR or SUSD4 with endo−/lysosomal markers quantified in BT-20 cells (C) and MDA-MB-468 cells (D) (Every data point represents the average of at least 10 cells per sample). All results were repeated in at least three independent biological experiments. Pearson’s correlation coefficient r where a minimal r value of 0.5 (dotted line) indicates significant colocalization

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Sushi domain-containing protein 4 binds to epithelial growth factor receptor and initiates autophagy in an EGFR phosphorylation independent manner

    doi: 10.1186/s13046-022-02565-1

    Figure Lengend Snippet: SUSD4 colocalization with endo−/lysosomal markers. Representative merged confocal images showing EGFR (red), SUSD4 (green), endo−/lysosomal markers (blue) and DAPI (magenta) in BT-20 cells (A) or MDA-MB-468 cells (B) expressing SUSD4 and corresponding mock control cells. Note that the combination of the colors red, green, and blue is presented in white color. Colocalization coefficient for EGFR or SUSD4 with endo−/lysosomal markers quantified in BT-20 cells (C) and MDA-MB-468 cells (D) (Every data point represents the average of at least 10 cells per sample). All results were repeated in at least three independent biological experiments. Pearson’s correlation coefficient r where a minimal r value of 0.5 (dotted line) indicates significant colocalization

    Article Snippet: Breast cancer cell lines BT-20 (ATCC), HS-578 T and MDA-MB-468 (DSMZ) were obtained directly from vendors and maintained in culture for a limited number of passages.

    Techniques: Expressing

    Effect of Tan IIA or STS on the growth of BT-20 breast cancer cells. (a) BT-20 cells were treated with 2, 10, 25, and 50 μ g/mL of Tan IIA or STS for 72 h. The cell growth was determined by WST-1 assay. (b) BT-20 cells were treated with 2, 10, 25, and 50 μ g/mL of Tan IIA or STS for 24 h or 48 h. The ratio of apoptosis was determined by flow cytometry. Data are the mean ± SD of triplicates from a representative assay of three separate experiments.

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: Effect of Supplementation of Tanshinone IIA and Sodium Tanshinone IIA Sulfonate on the Anticancer Effect of Epirubicin: An In Vitro Study

    doi: 10.1155/2011/841564

    Figure Lengend Snippet: Effect of Tan IIA or STS on the growth of BT-20 breast cancer cells. (a) BT-20 cells were treated with 2, 10, 25, and 50 μ g/mL of Tan IIA or STS for 72 h. The cell growth was determined by WST-1 assay. (b) BT-20 cells were treated with 2, 10, 25, and 50 μ g/mL of Tan IIA or STS for 24 h or 48 h. The ratio of apoptosis was determined by flow cytometry. Data are the mean ± SD of triplicates from a representative assay of three separate experiments.

    Article Snippet: The human breast cancer cell line BT-20 was obtained from the American Type Culture Collection (ATCC).

    Techniques: WST-1 Assay, Flow Cytometry

    Effect of Tan IIA or STS on epirubicin-induced cytotoxicity and apoptosis in BT-20 breast cancer cells. (a) BT-20 breast cancer cells were treated with 0–2 μ g/mL epirubicin in the presence or absence of STS (0–50 μ g/mL) or Tan IIA (0–50 μ g/mL) for 72 h. The cell growth was determined by WST-1 assay. (b) BT-20 cells were treated with 1 μ g/mL of epirubicin. The change in apoptosis with the addition of STS (50 μ g/mL) and Tan IIA (2, 10, 25, 50 μ g/mL) in epirubicin- (Epi-) treated BT-20 cells was determined by flow cytometry at 48 or 72 h. * P < .05 when compared with the control group; + P < .05 when compared with the epirubicin group. Data are the mean ± SD of triplicates from a representative assay of three separate experiments.

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: Effect of Supplementation of Tanshinone IIA and Sodium Tanshinone IIA Sulfonate on the Anticancer Effect of Epirubicin: An In Vitro Study

    doi: 10.1155/2011/841564

    Figure Lengend Snippet: Effect of Tan IIA or STS on epirubicin-induced cytotoxicity and apoptosis in BT-20 breast cancer cells. (a) BT-20 breast cancer cells were treated with 0–2 μ g/mL epirubicin in the presence or absence of STS (0–50 μ g/mL) or Tan IIA (0–50 μ g/mL) for 72 h. The cell growth was determined by WST-1 assay. (b) BT-20 cells were treated with 1 μ g/mL of epirubicin. The change in apoptosis with the addition of STS (50 μ g/mL) and Tan IIA (2, 10, 25, 50 μ g/mL) in epirubicin- (Epi-) treated BT-20 cells was determined by flow cytometry at 48 or 72 h. * P < .05 when compared with the control group; + P < .05 when compared with the epirubicin group. Data are the mean ± SD of triplicates from a representative assay of three separate experiments.

    Article Snippet: The human breast cancer cell line BT-20 was obtained from the American Type Culture Collection (ATCC).

    Techniques: WST-1 Assay, Flow Cytometry

    Uptake of epirubicin in BT-20 breast cancer cells in the presence or absence of STS (a) or Tan IIA (b) treatment for 24 h. Data are the mean ± SD of triplicates from a representative assay of three separate experiments. * P < .05 as compared with 1 μ g/mL epirubicin-treated BT-20 cells.

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: Effect of Supplementation of Tanshinone IIA and Sodium Tanshinone IIA Sulfonate on the Anticancer Effect of Epirubicin: An In Vitro Study

    doi: 10.1155/2011/841564

    Figure Lengend Snippet: Uptake of epirubicin in BT-20 breast cancer cells in the presence or absence of STS (a) or Tan IIA (b) treatment for 24 h. Data are the mean ± SD of triplicates from a representative assay of three separate experiments. * P < .05 as compared with 1 μ g/mL epirubicin-treated BT-20 cells.

    Article Snippet: The human breast cancer cell line BT-20 was obtained from the American Type Culture Collection (ATCC).

    Techniques:

    Akt-related pathway activation in the presence or absence of STS (or Tan IIA) in epirubicin-treated BT-20 breast cancer cells for 24 h. (a) Representative results of Western blotting showing changes in the levels of Akt, phospho-Akt, JNK, phospho-JNK, P38, phospho-p38, MAPK, and phospho-MAPK in BT-20 cells treated with STS and/or 1 μ g/mL epirubicin. (b) Representative results of Western blotting showing changes in the levels of Akt, phospho-Akt, JNK, phospho-JNK, P38, phospho-p38, MAPK, and phospho-MAPK in BT-20 cells treated with Tan IIA and/or 1 μ g/mL epirubicin.

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: Effect of Supplementation of Tanshinone IIA and Sodium Tanshinone IIA Sulfonate on the Anticancer Effect of Epirubicin: An In Vitro Study

    doi: 10.1155/2011/841564

    Figure Lengend Snippet: Akt-related pathway activation in the presence or absence of STS (or Tan IIA) in epirubicin-treated BT-20 breast cancer cells for 24 h. (a) Representative results of Western blotting showing changes in the levels of Akt, phospho-Akt, JNK, phospho-JNK, P38, phospho-p38, MAPK, and phospho-MAPK in BT-20 cells treated with STS and/or 1 μ g/mL epirubicin. (b) Representative results of Western blotting showing changes in the levels of Akt, phospho-Akt, JNK, phospho-JNK, P38, phospho-p38, MAPK, and phospho-MAPK in BT-20 cells treated with Tan IIA and/or 1 μ g/mL epirubicin.

    Article Snippet: The human breast cancer cell line BT-20 was obtained from the American Type Culture Collection (ATCC).

    Techniques: Activation Assay, Western Blot