Journal: Oncogene

Article Title: Induction of cells with cancer stem cell properties from nontumorigenic human mammary epithelial cells by defined reprogramming factors

doi: 10.1038/onc.2012.614

Figure Lengend Snippet: Reprogramming of human MCF-10A mammary epithelial cells. (a) Experimental scheme for the reprogramming of MCF-10A cells. (b) Phase-contrast images and immunofluorescence images of iPS-like colonies from MCF-10A cells (iPSL-10A) and normal human iPSCs stained with antibodies against OCT4, SOX2, TRA-1-60 and Nanog. Scale bar, 500 µm. (c) Semiquantitative reverse transcriptase–PCR (RT–PCR) analysis of iPSC markers in iPSL-10A cell clones 1–4, normal human iPSCs and MCF-10A cells. SOX2 and OCT4 are endogenously derived. (d) Immunoblotting of the stem cell marker proteins in iPSL-10A cell clones 1–4, normal human iPSCs and MCF-10A cells. (e) DNA methylation ‘heat map’ of iPSL-10A cells. DNA methylation analysis was performed using an Illumina Human Methylation 27 Beads Chip (MBL) with genomic DNA extracted from iPSL-10A clones 1 and 2, normal human iPSCs and MCF-10A cells. The β-value was calculated by a quantitative measure of the DNA methylation levels at specific CpG islands. Average β-values were subjected to unsupervised hierarchical clustering based on the Manhattan distance and average linkage. (f) High-resolution insertion-site analysis by linear amplification-mediated PCR (LAM PCR). Genomic DNA was prepared using phenol/chloroform extraction and subjected to LAM PCR. Amplicons were validated by sequencing. (g) Standard G-band chromosome analysis of MCF-10A and iPSL-10A cells. Arrows indicate identifiable aberrations common to both cell types.

Article Snippet: Human primary mammary epithelial cells were purchased from Kurabo Industrial (Osaka, Japan).

Techniques: Immunofluorescence, Staining, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Clone Assay, Derivative Assay, Western Blot, Marker, DNA Methylation Assay, Methylation, Amplification, Extraction, Sequencing

Journal: Oncogene

Article Title: Induction of cells with cancer stem cell properties from nontumorigenic human mammary epithelial cells by defined reprogramming factors

doi: 10.1038/onc.2012.614

Figure Lengend Snippet: Characterization of the CSC properties of iCSCL-10A clones. (a) Flow cytometric analysis of CD44 and CD24 expression in the MCF-10A, iCSCL-10A and MCF7 cell lines. The numbers indicate the percentage of each sub-population according to the CD44/CD24 expression profile. (b, c) Tumor sphere formation assays of MCF-10A-Ras, iCSCL-10A and MCF7 cell lines. Phase-contrast images of tumor spheres are shown (b). Values represent the mean ± s.e.m. (n=3, c). (d) Semiquantitative reverse transcriptase–PCR (RT–PCR) analysis of the expression of CSC- or epithelial-to-mesenchymal transition (EMT)-related genes. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was analyzed as a control. (e) Viability of MCF-10A-Ras, iCSCL-10A and MCF7 cell lines treated with various chemotherapeutic agents for 72 h by MTT assay. Values represent the mean ± s.e.m. (n=3). (f, g) iCSCL-10A and parental MCF-10A cells were treated with Juglone (5 µm) for 24 h and subjected to TUNEL (terminal deoxyribonucleotidyl transferase-mediated dUTP nick end-labeling) assay (f, brown color). TUNEL-positive cells were scored from triplicate independent experiments (g). Values represent the mean ± s.e.m. (n=3).

Article Snippet: Human primary mammary epithelial cells were purchased from Kurabo Industrial (Osaka, Japan).

Techniques: Clone Assay, Expressing, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Control, MTT Assay, TUNEL Assay, End Labeling

Journal: Oncogene

Article Title: Induction of cells with cancer stem cell properties from nontumorigenic human mammary epithelial cells by defined reprogramming factors

doi: 10.1038/onc.2012.614

Figure Lengend Snippet: iCSCL-10A cells form hierarchically organized tumors in vivo. (a) Tumor-seeding ability of iCSCL-10A, MCF-10A-Ras parental MCF-10A cells and iPSC-EBD. The indicated numbers of each cell type were injected into immunocompromised mice. The tumor-initiation ability per injection was then monitored. (b) Hematoxylin and eosin (H&E) staining of primary tumor tissues. Scale bar, 500 µm. (c) Immunohistochemical analysis of primary tumor tissues derived from iCSCL-10A cells using antibodies targeting hCD34 (endothelial), smooth muscle actin (SMA; myoblastic), β3-tubulin (neural), cytokeratin (CAM5.2, epithelial), vimentin (mesenchymal) and osteopontin (osteoblastic). Scale bar, 500 µm. (d) Immunofluorescent analysis with antibodies targeting SOX2 and cytokeratin (AE1/AE3). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar, 500 µm.

Article Snippet: Human primary mammary epithelial cells were purchased from Kurabo Industrial (Osaka, Japan).

Techniques: In Vivo, Injection, Staining, Immunohistochemical staining, Derivative Assay

Journal: Cancer research

Article Title: SIRT1 is essential for oncogenic signaling by estrogen/estrogen receptor ? in breast cancer

doi: 10.1158/0008-5472.CAN-11-1446

Figure Lengend Snippet: (A) SIRT1 was immunoprecipitated (IP) from ER− (HMEC, MCF10A, MB231 and MB453) and ER+ (MCF7 and ZR75.1) cells using SIRT1 or ERα antibodies. Immunoblotting (IB) was done with SIRT1 or ERα antibodies. (B) Colocalization of SIRT1 and ERα in MCF-7 and ZR75.1 cells. Hoechst staining was used to locate the nucleus. The scale bars represent 20 µm. (C) ER− MCF10A and MB231 cells were transfected with an expression construct of ERα. IP and IB were performed 48 h post-transfection with SIRT1 and ERα antibodies. (D) MCF10A cells were transfected with an ERα expression construct, and colocalization (arrows) was monitored with SIRT1 and ERα antibodies.

Article Snippet: Cell lines, plasmids and transfection The human normal mammary epithelial cell line HMEC was obtained from the Lonza, Walkersville, MD while the other two human normal mammary epithelial cell lines, MCF10A and MCF12A, were obtained from ATCC, Manassas, VA. HBL100, also a human normal mammary epithelial cell line, was kindly provided by Dr. S. Sukumar, Johns Hopkins University, Baltimore, MD.

Techniques: Immunoprecipitation, Western Blot, Staining, Transfection, Expressing, Construct