Journal: Stem Cells International

Article Title: Dexamethasone Provides Effective Immunosuppression for Improved Survival of Retinal Organoids after Epiretinal Transplantation

doi: 10.1155/2019/7148032

Figure Lengend Snippet: Primers.

Article Snippet: Antibodies against the following markers were used as primary antibodies for immunofluorescence microscopy at the dilutions indicated in parentheses: Ki67 (1 : 100, A11390, ABclonal, China), CHX10 (1 : 200, AB9016, Millipore, Germany), Brn3 (1 : 200, SC-6026X, Santa Cruz, USA), Islet1 (1 : 20, BM44446, Boster, China), HuD (1 : 100, SC-48421, Santa Cruz), RBPMS (1 : 100, ab152101, Abcam), β III-tubulin (1 : 200, ab7751, Abcam), neurofilament light polypeptide (NEFL; 1 : 100, A0257, ABclonal), microtubule-associated protein 2 (MAP2, 1 : 100, BM1243, Boster), vimentin (1 : 50, BM4029, Boster), glial fibrillary acidic protein (GFAP; 1 : 500, 3670S, Cell Signaling Technology, USA), CD68 (1 : 200, ab201340, Abcam), Iba1 (1 : 200, ab5076, Abcam), and SC121 (1 : 500, Y40410, Takara Bio).

Techniques:

Journal: Stem Cells International

Article Title: Dexamethasone Provides Effective Immunosuppression for Improved Survival of Retinal Organoids after Epiretinal Transplantation

doi: 10.1155/2019/7148032

Figure Lengend Snippet: Characteristics of retinal organoids after 30-45 days of induction. The frozen sections of retinal organoids were identified by immunofluorescence staining. (a, b) At this time, most cells of the organoids were differentiating towards RGCs; they were Islet1-, Brn3- and HuD-positive. (c) But some cells outside were still retinal progenitors; they were CHX10-positive. (d, e) And in suspension cultivation, little axons were developed.

Article Snippet: Antibodies against the following markers were used as primary antibodies for immunofluorescence microscopy at the dilutions indicated in parentheses: Ki67 (1 : 100, A11390, ABclonal, China), CHX10 (1 : 200, AB9016, Millipore, Germany), Brn3 (1 : 200, SC-6026X, Santa Cruz, USA), Islet1 (1 : 20, BM44446, Boster, China), HuD (1 : 100, SC-48421, Santa Cruz), RBPMS (1 : 100, ab152101, Abcam), β III-tubulin (1 : 200, ab7751, Abcam), neurofilament light polypeptide (NEFL; 1 : 100, A0257, ABclonal), microtubule-associated protein 2 (MAP2, 1 : 100, BM1243, Boster), vimentin (1 : 50, BM4029, Boster), glial fibrillary acidic protein (GFAP; 1 : 500, 3670S, Cell Signaling Technology, USA), CD68 (1 : 200, ab201340, Abcam), Iba1 (1 : 200, ab5076, Abcam), and SC121 (1 : 500, Y40410, Takara Bio).

Techniques: Immunofluorescence, Staining, Suspension

Journal: Stem Cells International

Article Title: Dexamethasone Provides Effective Immunosuppression for Improved Survival of Retinal Organoids after Epiretinal Transplantation

doi: 10.1155/2019/7148032

Figure Lengend Snippet: Comparisons of different marker expression profiles among the control, rapamycin- (RAP-) treated, and dexamethasone- (DEX-) treated groups. Two weeks after immunosuppressant treatment, (a) cells in the retinal organoids treated with RAP expressed higher levels of the retinal ganglion cell (RGC) markers Atoh7, Islet1, and Brn3b, the dendrite marker MAP2, the retinal progenitor marker PAX6 and CHX10, and photoreceptor marker CRX, compared to levels in the control group. These cells also expressed lower levels of the axon markers TUBB3 and NEFL than cells in the control group. In DEX-treatment group cells, dendrite, retinal progenitor, and photoreceptor markers (MAP2, CHX10, and CRX) were upregulated compared to levels in the control group. (b) However, 4 weeks after immunosuppressant treatment, both RAP- and DEX-treated cells showed higher expression levels of only PAX6 and CRX as compared to those in the control group. (c) Representative histogram of flow cytometry results. Each wave represented one group; the proportion of each group was calculated by the Blk group. There were 79.87 ± 5.92% of the RGCs in the control group, 60.97 ± 7.36% in the RAP group, and 63.6 ± 10.35% in the DEX group. CTRL: control group; RAP: rapamycin-treatment group; DEX: dexamethasone-treatment group; iPSCs: negative control group in RT-PCR; Blk: blank control group in flow cytometry.

Article Snippet: Antibodies against the following markers were used as primary antibodies for immunofluorescence microscopy at the dilutions indicated in parentheses: Ki67 (1 : 100, A11390, ABclonal, China), CHX10 (1 : 200, AB9016, Millipore, Germany), Brn3 (1 : 200, SC-6026X, Santa Cruz, USA), Islet1 (1 : 20, BM44446, Boster, China), HuD (1 : 100, SC-48421, Santa Cruz), RBPMS (1 : 100, ab152101, Abcam), β III-tubulin (1 : 200, ab7751, Abcam), neurofilament light polypeptide (NEFL; 1 : 100, A0257, ABclonal), microtubule-associated protein 2 (MAP2, 1 : 100, BM1243, Boster), vimentin (1 : 50, BM4029, Boster), glial fibrillary acidic protein (GFAP; 1 : 500, 3670S, Cell Signaling Technology, USA), CD68 (1 : 200, ab201340, Abcam), Iba1 (1 : 200, ab5076, Abcam), and SC121 (1 : 500, Y40410, Takara Bio).

Techniques: Marker, Expressing, Control, Flow Cytometry, Negative Control, Reverse Transcription Polymerase Chain Reaction

Journal: Nature communications

Article Title: DNMT1 is essential for mammary and cancer stem cell maintenance and tumorigenesis

doi: 10.1038/ncomms7910

Figure Lengend Snippet: a , Genome-scale distribution of DMRs identified in mammospheres of normal mammary stem cells and tumorospheres of tumor-initiating cells prepared from MMTV-Neu-Tg mice (n=3 mice in each). b , Cluster analysis of top 100 hypermethylated and few hypomethylated genes. c, Genome browser shot showing ChIP-Seq tracks for H3K27me3 (red) and H3K4me3 (green) levels near Isl1 in embryonic stem cells (ESCs), MaSCs, and luminal progenitors (LPs). d , UCSC genome browser screenshot showing DNA methylation profile at ISL1 locus in mammospheres and tumorospheres. Red and green colors indicate methylated and unmethylated CpG sites, respectively.

Article Snippet: For isolation of stem/progenitor cells, the following antibodies were used: CD49f and CD24 (Stem cell technology, eBioscience), CD45 and CD31 (BD bioscience), CD44 and ISL1 (BD bioscience), and Ter119 and CD61 (eBioscience).

Techniques: ChIP-sequencing, DNA Methylation Assay, Methylation

Journal: Nature communications

Article Title: DNMT1 is essential for mammary and cancer stem cell maintenance and tumorigenesis

doi: 10.1038/ncomms7910

Figure Lengend Snippet: a , ISL1 promoter was analyzed from TCGA human methylation 27K methylation dataset at a single CpG site (cg21410991) for differential methylation in luminal A, luminal B, HER2-Neu, and basal breast cancer subtypes. b , ISL1 expression in ER-positive (n=12) and negative (n=4) breast tumor and respective normal breast tissues show reduced expression of this gene in both tumor subtypes. c , ISL1 expression in human non-transformed normal mammary epithelial and human breast cancer cell lines was analyzed by qPCR and western blot analysis. d , Representative FACS data confirming exogenous expression of ISL1 in CAL51 cell line. e , Representative images of colony formation assay of CAL51-pCDH and CAL51-ISL1 stable cell lines show that ISL1 expression significantly reduces colony formation. The resulting colonies were stained with Giemsa dye and bound Giemsa dye were dissolved and quantified by spectrophotometer analysis. Data represents mean ± SD from three independent experiments. f , Histogram shows that ISL1 expression in CAL51 cells increased apoptosis (Annexin V + /PI − ) compared to vector control. Data are mean ± SEM of 3 independent experiments. g , CAL51-pCDH and CAL51-ISL1 expressing stable cells were subjected to in vitro scratch assay and images were captured using phase-contrast microscope at 12 h after incubation. Scale bar 200 µm. h , Representative FACS plot shows decreased cancer stem cells (CD44 + CD24 − ) in ISL1 expressing CAL51 cells. i , Histogram showing percent decrease in cancer stem cells after ISL1 expression in CAL51 cells. j , Relative ISL1 expression in wild-type and DNMT1 -null mice after 3D and 2D culture. (n=3 mice). Statistical analysis was performed using unpaired Student's t -tests.

Article Snippet: For isolation of stem/progenitor cells, the following antibodies were used: CD49f and CD24 (Stem cell technology, eBioscience), CD45 and CD31 (BD bioscience), CD44 and ISL1 (BD bioscience), and Ter119 and CD61 (eBioscience).

Techniques: Methylation, Expressing, Transformation Assay, Western Blot, Colony Assay, Stable Transfection, Staining, Spectrophotometry, Plasmid Preparation, In Vitro, Wound Healing Assay, Microscopy, Incubation

Journal: Nature communications

Article Title: DNMT1 is essential for mammary and cancer stem cell maintenance and tumorigenesis

doi: 10.1038/ncomms7910

Figure Lengend Snippet: Kaplan-Meier plots of overall survival of breast cancer patients in whole data sets for all breast cancer patients ( a ), estrogen receptor positive and progesterone receptor positive patients ( b ), and estrogen receptor negative patients ( c ), stratified by ISL1 expression. Data were obtained from the Kaplan-Meier plotter breast cancer survival analysis database. The p value was calculated using a log rank test.

Article Snippet: For isolation of stem/progenitor cells, the following antibodies were used: CD49f and CD24 (Stem cell technology, eBioscience), CD45 and CD31 (BD bioscience), CD44 and ISL1 (BD bioscience), and Ter119 and CD61 (eBioscience).

Techniques: Expressing