Journal: Oncotarget

Article Title: MicroRNA-1 down-regulates proliferation and migration of breast cancer stem cells by inhibiting the Wnt/β-catenin pathway

doi:

Figure Lengend Snippet: MCF-7 and SKBR3 cells were transfected with, or without, miR-1 NC, miR-1 inhibitor, or miR-1 mimic for 24 or 48 h. The relative levels of miR-1 expression in different groups of cells were determined by quantitative RT-PCR A. The frequency of CD44+CD24− CSCs was determined by flow cytometry B–C. Data are representative FACS charts and expressed as the mean ± SD of each group of cells from three separate experiments. * p < 0.05, ** p < 0.01 vs. the control CSCs.

Article Snippet: 293T cells and breast carcinoma cell lines, MCF-7 (lumina A, ER+, PR+, HER2-), MDA-MB-231 (basal-like, ER-, PR-, HER2-) and SKBR3 (ER-, PR-, Her-2+), were obtained from the American Type Culture Collection (ATCC, Manassas, USA) and cultured in 10% fetal bovine serum (FBS) DMEM medium (complete medium, Invitrogen, Grand island, USA) at 37°C in an atmosphere of 5% CO 2 .

Techniques: Transfection, Expressing, Quantitative RT-PCR, Flow Cytometry, Control

Journal: Oncotarget

Article Title: MicroRNA-1 down-regulates proliferation and migration of breast cancer stem cells by inhibiting the Wnt/β-catenin pathway

doi:

Figure Lengend Snippet: 293T cells were transfected with luciferase reporter plasmids containing the 3UTR sequences of the fizzled 4, 5, 7, or TNKS2 and miR-1 or control miR-1NC and the effect of miR-1 on luciferase activity was determined A. The luciferase activity in the control cells that had been transfected with miR-1NC were considered as 100%. CD44+CD24− CSC from MCF-7 and SKBR3 cells were sorted and transfected with, or without, miR-1NC, miR-1inhibitor, or miR-1mimic for 24 or 48 h. The relative levels of fizzled 7, TNKS2 and c-Myc to control GAPDH were determined by Western blot B. In addition, the relative levels of cytoplasmic and nuclear β-catenin to GAPDH or lumen B1 in each group of cells were determined by Western blot C. Moreover, MCF-7/CSC and SKBR3/CSC were transfected with miR-1mimic, miR-1NC or miR-1inhibitor for 24 h. The cells in each group were transfected with TopFlash and pRL-TK for 48 h. The relative levels of β-catenin-dependent firefly luciferase activity in different groups of CSCs were determined by the dual luciferase assay D. The relative levels of β-catenin-dependent luciferase activity in the control cells that had been transfected with miR-1NC, TopFlash and pRL-TK were designated as 1. Finally, the relative levels of Oct4 and Nanog expression in each group of cells were determined by Western blot E. Data are representative images and expressed as the mean ± SD of each group of cells from three separate experiments. * p < 0.05; ** p < 0.01.

Article Snippet: 293T cells and breast carcinoma cell lines, MCF-7 (lumina A, ER+, PR+, HER2-), MDA-MB-231 (basal-like, ER-, PR-, HER2-) and SKBR3 (ER-, PR-, Her-2+), were obtained from the American Type Culture Collection (ATCC, Manassas, USA) and cultured in 10% fetal bovine serum (FBS) DMEM medium (complete medium, Invitrogen, Grand island, USA) at 37°C in an atmosphere of 5% CO 2 .

Techniques: Transfection, Luciferase, Control, Activity Assay, Western Blot, Expressing

Journal: Oncotarget

Article Title: MicroRNA-1 down-regulates proliferation and migration of breast cancer stem cells by inhibiting the Wnt/β-catenin pathway

doi:

Figure Lengend Snippet: CD44+CD24− CSCs from MCF-7 and SKBR3 cells were transfected with, or without, miR-1 NC, miR-1 inhibitor, or miR-1 mimic for 24 or 48 h. Their proliferation was determined by MTT assay. Data are expressed as the mean rates ± SD of each group of cells from three separate experiments and proliferation of untransfected control CSCs was designated as 1. * p < 0.05; ** p < 0.01 vs. the miR-1 NC and control CSCs.

Article Snippet: 293T cells and breast carcinoma cell lines, MCF-7 (lumina A, ER+, PR+, HER2-), MDA-MB-231 (basal-like, ER-, PR-, HER2-) and SKBR3 (ER-, PR-, Her-2+), were obtained from the American Type Culture Collection (ATCC, Manassas, USA) and cultured in 10% fetal bovine serum (FBS) DMEM medium (complete medium, Invitrogen, Grand island, USA) at 37°C in an atmosphere of 5% CO 2 .

Techniques: Transfection, MTT Assay, Control

Journal: Oncotarget

Article Title: MicroRNA-1 down-regulates proliferation and migration of breast cancer stem cells by inhibiting the Wnt/β-catenin pathway

doi:

Figure Lengend Snippet: CD44+CD24− CSC from MCF-7 and SKBR3 cells were transfected with, or without, miR-1 NC, miR-1 inhibitor, or miR-1 mimic. Four hours after transfection, the migration of different groups of CSC for the indicated time points was determined by transwell migration assay A. In addition, two days after transfection, the wound healing of different groups of cells within 24 or 48 h was tested B. Data are representative images and expressed as the mean ± SD of each group of cells from three separate experiments. The number of migrated SKBR3/CSCs was similar to those of SKBR3/miR-1NC CSCs and the migration distance of control MCF-7CSC and SKBR3/CSC was similar to those of MCF-7/miR-1NC and SKBR3/miR-1NC CSCs, respectively (data not shown). * p < 0.05; ** p < 0.01 vs. the miR-1 NC-transfected CSCs.

Article Snippet: 293T cells and breast carcinoma cell lines, MCF-7 (lumina A, ER+, PR+, HER2-), MDA-MB-231 (basal-like, ER-, PR-, HER2-) and SKBR3 (ER-, PR-, Her-2+), were obtained from the American Type Culture Collection (ATCC, Manassas, USA) and cultured in 10% fetal bovine serum (FBS) DMEM medium (complete medium, Invitrogen, Grand island, USA) at 37°C in an atmosphere of 5% CO 2 .

Techniques: Transfection, Migration, Transwell Migration Assay, Control

Journal: PLoS ONE

Article Title: 13-amino derivatives of dehydrocostus lactone display greatly enhanced selective toxicity against breast cancer cells and improved binding energies to protein kinases in silico

doi: 10.1371/journal.pone.0271389

Figure Lengend Snippet: Anticancer activities of 13-amino derivatives compounds against HCC70, MCF-7, and MCF12A cell lines.

Article Snippet: The cancer cells MCF-7 hormone receptor positive breast cancer [estrogen receptor (ER) + , progesterone receptor (PR) + , human epidermal growth factor-2 (HER-2) -; ATCC: HTB-22), HCC70 triple negative breast cancer (TNBC, ER - , PR - , HER-2 - ; ATCC: CRL-2315) and a non-tumorigenic breast epithelial cell line MCF-12A (ATCC: CRL-10782), were seeded at a density of 5000 cells/well in a 96 well plate and were allowed to adhere overnight at 37°C in a 9% CO 2 incubator.

Techniques:

Journal: BMC Cancer

Article Title: Vasculogenic mimicry-associated ultrastructural findings in human and canine inflammatory breast cancer cell lines

doi: 10.1186/s12885-019-5955-z

Figure Lengend Snippet: Transmission electron microscopy of SUM149 ( a , b ) and IPC-366 ( c ) in adherent conditions (2D). Large individualized round cells showing cytoplasmic membrane processes (microvilli) and marked anisocaryosis and anisocytosis and prominent nucleoli. d , e IPC-366. Peroxisomes (arrow) in close apposition to lipid droplets (asterisks). Original magnification; a , b × 6,000, c × 4,000, d × 12,000, e × 50,000

Article Snippet: SUM149 triple negative (ER−, PR−, HER-2−) human inflammatory breast carcinoma cell line was obtained from Asterand, Plc. (Detroit, Michigan, USA) in 2015, was maintained in Ham’s F-12 media supplemented with 10% fetal bovine serum (FBS) (Sigma Aldrich, Madrid, Spain),1 μg mL −1 hydrocortisone, 5 μg mL −1 insulin and 1% penicillin–streptomycin solution and 1% amphotericin B (Sigma Aldrich, Madrid, Spain).

Techniques: Transmission Assay, Electron Microscopy, Membrane

Journal: BMC Cancer

Article Title: Vasculogenic mimicry-associated ultrastructural findings in human and canine inflammatory breast cancer cell lines

doi: 10.1186/s12885-019-5955-z

Figure Lengend Snippet: Transmission electron microscopy of IPC-366 ( a , b ) in adherent conditions (2D) and SUM149 ( c , d , e , f ) in non-adherent conditions (mammospheres). a and b : Endothelial-like cells (ELCs) in formation. Multiple empty cytoplasmic spaces (arrows), with microvilli covered by cytoplasmic membrane (insert) and nucleus margination. c , d and e : ELCs showing the characteristic morphology: a unique cytoplasmic empty space and eccentric nucleus. f : ELC cytoplasm with Weibel-Palade bodies (arrows). Original magnification; a , d ) × 6,000, b ) × 10,000, c ) × 3,000, e ) × 4,000, f ) × 60,000

Article Snippet: SUM149 triple negative (ER−, PR−, HER-2−) human inflammatory breast carcinoma cell line was obtained from Asterand, Plc. (Detroit, Michigan, USA) in 2015, was maintained in Ham’s F-12 media supplemented with 10% fetal bovine serum (FBS) (Sigma Aldrich, Madrid, Spain),1 μg mL −1 hydrocortisone, 5 μg mL −1 insulin and 1% penicillin–streptomycin solution and 1% amphotericin B (Sigma Aldrich, Madrid, Spain).

Techniques: Transmission Assay, Electron Microscopy, Membrane

Journal: BMC Cancer

Article Title: Vasculogenic mimicry-associated ultrastructural findings in human and canine inflammatory breast cancer cell lines

doi: 10.1186/s12885-019-5955-z

Figure Lengend Snippet: Scanning electron microscopy of IPC-366 ( a , b , c , e ) and SUM149 ( d , f ) mammospheres. a Joint cells covered by numerous cytoplasmic projections (microvilli). b Magnification of microvilli. c and d Spherical and flattened cells, respectively. e and f Mammary acini-like structures

Article Snippet: SUM149 triple negative (ER−, PR−, HER-2−) human inflammatory breast carcinoma cell line was obtained from Asterand, Plc. (Detroit, Michigan, USA) in 2015, was maintained in Ham’s F-12 media supplemented with 10% fetal bovine serum (FBS) (Sigma Aldrich, Madrid, Spain),1 μg mL −1 hydrocortisone, 5 μg mL −1 insulin and 1% penicillin–streptomycin solution and 1% amphotericin B (Sigma Aldrich, Madrid, Spain).

Techniques: Electron Microscopy

Journal: Nature immunology

Article Title: St3gal1 and βII-spectrin pathways control CAR-T cell migration to target tumor sites

doi: 10.1038/s41590-023-01498-x

Figure Lengend Snippet: A. Gating strategy for detection of hHER2-CAR-T cells. A median of > 90% of mouse CD8 T cells transduced with the hHER2-CAR retrovirus were positive for the transgene as judged by flow cytometry analysis. B. B16F10 cells were transfected with mammalian human HER2 construct with lipofectamine. Cells were grown in the presence of G418 and sorted twice to generate single cell clones. C. CAR-T-mediated B16-HER2 cell killing assay. Apoptotic cells were stained for Annexin V and analyzed by flow cytometry. D. Whole mouse lung before (left) and after (right) CUBIC clearing.

Article Snippet: To detect CAR expression level on T cells, 1×10 6 CAR-T cells were pre-incubated with anti-mouse CD16/CD32 Fc blocker (1 μg/million cells in 100 μl, BD Bioscience) for 10 min at 4°C and incubated with recombinant human ErbB2/Her2 Fc chimera protein (R&D systems) at 4°C for 30 min. After washing, the cells were incubated with PE-anti-Human IgG Fc secondary antibody (eBioscience) or DyLight/650-anti-Human IgG Fc secondary antibody (Invitrogen) at 4°C for 30 min.

Techniques: In Vivo, Transduction, Flow Cytometry, Transfection, Construct, Clone Assay, Staining

Journal: Nature immunology

Article Title: St3gal1 and βII-spectrin pathways control CAR-T cell migration to target tumor sites

doi: 10.1038/s41590-023-01498-x

Figure Lengend Snippet: A. Generation of GFP-ROSAβII-spectrin mouse model (Rosa26tm(CAG-LSL-Sptbn1-IRES-GFP)). Mouse Sptbn1 cDNA was inserted into the CAG-STOP-GFP-Rosa targeting vector, CTV, between a floxed Stop cassette and the internal ribosome entry site (IRES) followed by the enhanced Green Fluorescent Protein gene (eGFP). Transcription is under control of the CAG promoter. The targeting vector contained Rosa26 homology arms (1 kb 5′ and 3.8 kb 3′), so that the entire loxP-stop-loxP-Tmc2-IRES-GFP transcriptional cassette was inserted into the first intron of Rosa26 gene on chromosome 6. B & C. Flow cytometry analysis of IFNγ and TNFα expression in hHER2-CAR transfected T cells from GFP-ROSAβII-spectrin mouse (TβII-spectrin) after treated with PBS or Tat-Cre Recombinase. Cells were co-cultured with B16-HER2 cells. Data represent mean ± SEM. n = 9. D. CAR-mediated B16-HER2 cell killing assay with TβII-spectrin cells after treated with PBS or Tat-Cre Recombinase. Cell death was stained for NucSpot. Data represent mean ± SEM. n = 9. E. Activated CD4+ T cell (OT-II) migration on ICAM-1 coated plates ± CXCL12. Cells were treated with PTx (6 hr) or Gallein (30 min) where indicated. Data were collected from 2 independent experiments (n = 2, 17–34 individual cells per mouse). Data represent mean ± SEM Statistical analyses were performed using one-way ANOVA with Bonferroni post-test. *P = 0.007. F. The pie charts depict the proportion of CD4 T cells distributed in the tumour, blood, LN/spleen, or lung/liver 72 h post-injection. G. Expression levels of βII-spectrin and St3gal1 in human CD4 and CD8 T cells (before and after activation). Loading control: β-actin. Representative western blot images from three independent experiments are shown. H. Expression levels of βII-spectrin in human CD8 memory T cells (CD8+CD45RO+CD45RA–CD56–CD57–). Loading control: β-actin. Representative western blot images from three independent experiments are shown.

Article Snippet: To detect CAR expression level on T cells, 1×10 6 CAR-T cells were pre-incubated with anti-mouse CD16/CD32 Fc blocker (1 μg/million cells in 100 μl, BD Bioscience) for 10 min at 4°C and incubated with recombinant human ErbB2/Her2 Fc chimera protein (R&D systems) at 4°C for 30 min. After washing, the cells were incubated with PE-anti-Human IgG Fc secondary antibody (eBioscience) or DyLight/650-anti-Human IgG Fc secondary antibody (Invitrogen) at 4°C for 30 min.

Techniques: Expressing, Plasmid Preparation, Control, Flow Cytometry, Transfection, Cell Culture, Staining, Migration, Injection, Activation Assay, Western Blot