Journal: PLoS ONE

Article Title: MicroRNAs Induce Epigenetic Reprogramming and Suppress Malignant Phenotypes of Human Colon Cancer Cells

doi: 10.1371/journal.pone.0127119

Figure Lengend Snippet: A) Scheme of cancer reprogramming methods by transfection of miR-302s or miR-302s plus miR-369s. B) miR-302s-induced morphological changes in HT29 cells at days 1, 6, and 28. Reprogrammed HT29 have an embryonic stem cells-like appearance. Scale bars, 100 μm. C) Cells stained using alkaline phosphatase (AP) live stain. Scale bars, 100 μm. D) Relative expression of OCT3/4 , SOX2 and NANOG compared to AP − cells by qRT-PCR (n = 3). E) Relative expression of miR-302s and miR-369s in AP + , AP − , and NTERA cells. Mean expression of each miR was compared to that in NTERA cells (n = 3). F) Immunofluorescence of pluripotent stem cell markers Oct3/4, Sox2, and Nanog. Nuclei were stained with DAPI. Scale bars, 100 μm (original magnification, ×200).

Article Snippet: Slides were heated in antigen retrieval buffer for 40 min, blocked with goat or horse serum for 20 min at room temperature, and incubated with monoclonal mouse anti-human Ki67 antigen (1:400, DAKO), polyclonal rabbit anti-human Oct3/4 (1:100, MBL), polyclonal rabbit anti-human Sox2 (1:200, MBL), or monoclonal mouse anti-human CK20 (DAKO) antibodies overnight at 4°C.

Techniques: Transfection, Staining, Expressing, Quantitative RT-PCR, Immunofluorescence

Journal: PLoS ONE

Article Title: MicroRNAs Induce Epigenetic Reprogramming and Suppress Malignant Phenotypes of Human Colon Cancer Cells

doi: 10.1371/journal.pone.0127119

Figure Lengend Snippet: A) Tumor-bearing mice were treated with systemic administration of carbonate apatite (CA)- complexed miR-302s, negative control (NC) miR, or CA only every second day, with a total of six injections. Tumor growth curves are shown. Asterisk denotes a p-value in the Mann-Whitney U-test of < 0.05. B) Tumor-bearing mice were treated with systemic administration of CA-equipped miR-302s plus miR-369s, NC miR, or CA only daily, with a total of 14 injections, similar to (A). C) Representative hematoxylin and eosin-stained images of tumor sections from the indicated experimental groups. Areas with denaturated nuclei were predominantly observed in xenografts treated with miR-302s/CA complexes and miR-302s plus miR-369s/CA complexes. Scale bars, 100 μm (magnification ×200). D) Confirmation of intratumoral apoptosis by TUNEL staining in tumor sections obtained from xenografts treated with CA-mediated miR-302s, miR-302s plus miR-369s, NC, or CA only. Scale bars, 100 μm (magnification ×200). E) Immunohistochemistry analysis of the proliferation marker Ki-67 in xenografts treated with miR-302s, miR-302s plus miR-369s, NC miR or the mock control. Representative images are shown. Scale bars, 100 μm (magnification ×200). Ki-67-positive cells were counted in three random fields from different three xenografts in each group. F) Relative expression of pluripotency-related markers OCT3/4, SOX2 and NANOG in the indicated experimental groups. Data are shown in comparison with the NC miR group. (n = 3). G) Representative immunohistochemical images of tumor sections from the indicated experiment groups. Oct3/4 and Sox2 were predominantly observed in xenografts treated with miR-302s and miR-302s plus miR-369s. Alcian blue staining showed the decrease of mucin production in these miR-treated-groups. CK20 expression was also decreased in xenografts treated with miR-302s plus miR-369s. Scale bars, 100 μm (magnification ×200).

Article Snippet: Slides were heated in antigen retrieval buffer for 40 min, blocked with goat or horse serum for 20 min at room temperature, and incubated with monoclonal mouse anti-human Ki67 antigen (1:400, DAKO), polyclonal rabbit anti-human Oct3/4 (1:100, MBL), polyclonal rabbit anti-human Sox2 (1:200, MBL), or monoclonal mouse anti-human CK20 (DAKO) antibodies overnight at 4°C.

Techniques: Negative Control, MANN-WHITNEY, Staining, TUNEL Assay, Immunohistochemistry, Marker, Expressing, Immunohistochemical staining