Journal: PLoS ONE

Article Title: Mbd3 , a Component of NuRD/Mi-2 Complex, Helps Maintain Pluripotency of Mouse Embryonic Stem Cells by Repressing Trophectoderm Differentiation

doi: 10.1371/journal.pone.0007684

Figure Lengend Snippet: A) Quantitative RT-PCR analysis Mbd3 mRNA expression in Mbd3 shRNA transfected CGR8 cells, which were selected with puromycin for five days. Gene expressions are normalized to internal control β -actin and presented as the fold induction relative to control shRNA. Error bars represent standard deviation from three technical repeats. B) Mbd3 shRNA transfections downregulates endogenous Mbd3 protein expression in CGR8 cells detected on western blots. Cells were transfected with indicated shRNA plasmids. C) Growth curves of CGR8 cells transfected with shRNA plasmids. Only GFP positive cells were counted. Error bars represent standard deviation. D) CGR8 cells were transfected either with control shRNA(a, a′) or Mbd3 shRNA1/2 (b, b′ and c, c′) and selected for five days with puromycin. GFP fluorescence in a′, b′, c′ indicate that the cells harbor shRNA plasmids. The scale bar represents 50μm.

Article Snippet: RNA interference (RNAi) target sequences for Mbd3 were selected using Ambion siRNA converter online software ( http://www.ambion.com/techlib/misc/siRNA_finder.html ).

Techniques: Quantitative RT-PCR, Expressing, shRNA, Transfection, Standard Deviation, Western Blot, Fluorescence

Journal: PLoS ONE

Article Title: Mbd3 , a Component of NuRD/Mi-2 Complex, Helps Maintain Pluripotency of Mouse Embryonic Stem Cells by Repressing Trophectoderm Differentiation

doi: 10.1371/journal.pone.0007684

Figure Lengend Snippet: Equal amounts of DNase-treated total RNA were subjected to quantitative RT-PCR analysis (A, B). A) Cells were transfected with shRNA plasmids and selected for six days with puromycin. B) Cells were co-transfected with a shRNA plasmid and human Mbd3 or control plasmid and selected with both puromycin and G418 for five days. The target sequences in Mbd3 shRNA1 and 2 do not exist in human Mbd3 sequence. C) Chromatin immunoprecipitation (ChIP) analysis using acetylated histone 3 antibody. CGR8 cells were transfected with shRNA plasmids and selected for five days with puromycin. Various pairs of PCR primer were designed to scan respective promoters. Gene expressions were normalized to internal control β -actin and presented as the fold induction relative to control samples. Error bars in panel A, B and C represent standard deviation from three technical repeats.

Article Snippet: RNA interference (RNAi) target sequences for Mbd3 were selected using Ambion siRNA converter online software ( http://www.ambion.com/techlib/misc/siRNA_finder.html ).

Techniques: Quantitative RT-PCR, Transfection, shRNA, Plasmid Preparation, Sequencing, Chromatin Immunoprecipitation, Standard Deviation

Journal: PLoS ONE

Article Title: Mbd3 , a Component of NuRD/Mi-2 Complex, Helps Maintain Pluripotency of Mouse Embryonic Stem Cells by Repressing Trophectoderm Differentiation

doi: 10.1371/journal.pone.0007684

Figure Lengend Snippet: A) Quantitative RT-PCR analysis of Cdx2 and Oct4 in Mbd3 knockdown stable lines. Control ESL represents control siRNA stably transfected mouse ES cells; ESL F5, ESL D3 ESL E8, ESL D8, and ESL G11 represent five independent Mdb3 siRNA stably transfected mouse ES cells. Error bars represent standard deviation from three technical repeats. B) Chimeric analysis of control and three independent siRNA ES cells in wild type mouse embryos. siRNA stably trasnfected cells were aggregated with wild type mouse embryos at the eight-cell stage and cultured to the blastocyst stage (a–l). in vitro morphology of the siRNA ES cells used in aggregations (m–p). Aggregated cells marked by DsRed. Scale bars: l, 200μm (also applies to a to k); p, 100μm (also applies to m, n, o).

Article Snippet: RNA interference (RNAi) target sequences for Mbd3 were selected using Ambion siRNA converter online software ( http://www.ambion.com/techlib/misc/siRNA_finder.html ).

Techniques: Quantitative RT-PCR, Stable Transfection, Transfection, Standard Deviation, Cell Culture, In Vitro

Journal: PLoS ONE

Article Title: Mbd3 , a Component of NuRD/Mi-2 Complex, Helps Maintain Pluripotency of Mouse Embryonic Stem Cells by Repressing Trophectoderm Differentiation

doi: 10.1371/journal.pone.0007684

Figure Lengend Snippet: Mouse ES cells transfected with contro or Mbd3 shRNAs for three days were cultured in trophoblast stem cell medium to promote differentiation into TS cells. GFP expression was used to monitor shRNA plasmids transfection (b, c, g, h, l, and m). Hoechst stains the cell nuclei. Red fluorescence shown on cell boundaries indicates the expression of cadherin 3, a surface antigen specific to trophoblast cells (d, e, i, j). Scale bars: l, 50μm (also applies to a, b, f, g, k); o, 50μm (also applies to c–e, h–j, m–n).

Article Snippet: RNA interference (RNAi) target sequences for Mbd3 were selected using Ambion siRNA converter online software ( http://www.ambion.com/techlib/misc/siRNA_finder.html ).

Techniques: Transfection, Cell Culture, Expressing, shRNA, Fluorescence