Journal: PLoS Genetics

Article Title: Two cis -regulatory SNPs upstream of ABCG2 synergistically cause the blue eggshell phenotype in the duck

doi: 10.1371/journal.pgen.1009119

Figure Lengend Snippet: (A) Supershift EMSA was performed using CTCF antibody. CTCF antibody (1 μg) was incubated with 0.5 μg recombinant human CTCF protein prior to the addition of the binding buffer and probe. G and A represent labelled probes containing the white eggshell and blue eggshell allele, respectively, and G cold and A cold represent cold probes containing the white eggshell and blue eggshell allele, respectively. (B) ChIP-qPCR analysis of CTCF binding on variation M5 in uteruses from blue-eggshelled and white-eggshelled ducks. Chromatin extracts from four individuals of each genotype were immunoprecipitated with CTCF antibody. ChIP-enriched DNA was quantified by qPCR with primers specific for variation M5. Rabbit IgG was used as a negative control. Values of immunoprecipitated samples were normalized to that of the input DNA. Data are presented as mean±SD from four independent individuals.

Article Snippet: CTCF antibody (1μg) was incubated with 0.5 μg recombinant human CTCF protein (Novus biologicals, Littleton, USA) prior to addition of the binding buffer and probe.

Techniques: Incubation, Recombinant, Binding Assay, ChIP-qPCR, Immunoprecipitation, Negative Control

Journal: Oncogene

Article Title: CTCF cooperates with noncoding RNA MYCNOS to promote neuroblastoma progression through facilitating MYCN expression.

doi: 10.1038/onc.2015.422

Figure Lengend Snippet: Figure 1. CTCF targets the binding sites within MYCN promoter to facilitate its expression in NB cells. (a) UCSC Genome Browser showing the enrichment of CTCF at bases −890/ −519 relative to the transcription start site (TSS) of MYCN in human embryonic stem cells (hESCs) and NB cell lines BE(2)-C and SK-N-SH. (b) Dual-luciferase assay showing the activity of MYCN promoter reporters (pE/E MYCN-Luc and pE/B MYCN-Luc) in SH-SY5Y, SK-N-SH, IMR32 and BE(2)-C cells stably transfected with empty vector (mock), CTCF, scramble shRNA (sh-Scb) or sh-CTCF (*Po0.01 vs mock or sh-Scb). (c) Dual-luciferase assay indicating the activity of MYCN promoter reporter (pE/E MYCN-Luc) with mutation of two CTCF binding sites, locating at bases −772/ −757 and −572/ −561 relative to TSS, in NB cells stably transfected with mock, CTCF, sh-Scb or sh-CTCF. (d, e) Real-time quantitative RT–PCR and western blot assays showing the expression of CTCF and MYCN in NB cells stably transfected with mock, CTCF, sh-Scb or sh-CTCF (*Po0.01 vs mock or sh-Scb).

Article Snippet: Immunohistochemistry Immunohistochemical staining and quantitative evaluation were performed as previously described,29–31 with antibodies specific for MYCN (sc22835) and CTCF (sc-271474, Santa Cruz Biotechnology; 1:200 dilutions).

Techniques: Binding Assay, Expressing, Luciferase, Activity Assay, Stable Transfection, Transfection, Plasmid Preparation, shRNA, Mutagenesis, Quantitative RT-PCR, Western Blot

Journal: Oncogene

Article Title: CTCF cooperates with noncoding RNA MYCNOS to promote neuroblastoma progression through facilitating MYCN expression.

doi: 10.1038/onc.2015.422

Figure Lengend Snippet: Figure 3. Functional interaction between CTCF and MYCNOS in regulating the MYCN expression in NB cells. (a) RIP assay revealing the interaction between MYCNOS and CTCF protein in SH-SY5Y cells transfected with a series of exon truncates of MYCNOS. (b) RNA EMSA showing the interaction between recombinant CTCF protein and biotin-labeled RNA probe for exons 1–3 of MYCNOS, with or without addition of unlabeled RNA probe. (c) Biotin-labeled RNA pull-down assay showing the interaction between MYCNOS and CTCF protein in BE(2)-C cells. (d) In vitro binding assay for the interaction between full-length (1–727 amino acids), amino-terminus (1–291 amino acids), zinc-finger domain (291–576 amino acids), zinc-fingers 9–11 (500–576 amino acids) or carboxyl-terminus (576–727 amino acids) of CTCF protein with MYCNOS. (e) Dual-luciferase assay indicating the activity of MYCN promoter reporter (pE/E MYCN-Luc) in NB cells stably transfected with empty vector (mock), MYCNOS or CTCF and co-transfected with scramble shRNA (sh-Scb), sh-CTCF or sh-MYCNOS, respectively (*Po0.01 vs mock+sh-Scb). (f) Western blot assay showing the expression of CTCF and MYCN in SH-SY5Y and BE(2)-C cells stably transfected with mock, MYCNOS or CTCF and co-transfected with sh-Scb, sh-CTCF or sh-MYCNOS, respectively.

Article Snippet: Immunohistochemistry Immunohistochemical staining and quantitative evaluation were performed as previously described,29–31 with antibodies specific for MYCN (sc22835) and CTCF (sc-271474, Santa Cruz Biotechnology; 1:200 dilutions).

Techniques: Functional Assay, Expressing, Transfection, Recombinant, Labeling, Pull Down Assay, In Vitro, Binding Assay, Luciferase, Activity Assay, Stable Transfection, Plasmid Preparation, shRNA, Western Blot

Journal: Oncogene

Article Title: CTCF cooperates with noncoding RNA MYCNOS to promote neuroblastoma progression through facilitating MYCN expression.

doi: 10.1038/onc.2015.422

Figure Lengend Snippet: Figure 4. MYCNOS facilitates the recruitment of CTCF and chromatin remodeling at MYCN promoter in NB cells. (a) UCSC Genome Browser showing the recruitment of RNA Pol II, active chromatin markers (H3K4me2 and H3K4me3) and repressive chromatin markers (H3K9me3 and H3K27me3) around CTCF binding sites. (b) ChIP and real-time quantitative PCR (qPCR) assays indicating the endogenous CTCF binding at −861/ −521 bp relative to MYCN transcription start site (TSS), and the changes in the binding of CTCF to MYCN promoter in BE(2)-C and SH-SY5Y cells stably transfected with scramble shRNA (sh-Scb), sh-MYCNOS, empty vector (mock) or MYCNOS (*Po0.01 vs sh-Scb or mock). (c) ChIP and real-time qPCR assays showing the binding of RNA Pol II, H3K4me2, H3K4me3, H3K9me3 and H3K27me3 to MYCN promoter in BE (2)-C cells stably transfected with mock or CTCF and co-transfected with sh-Scb or sh-MYCNOS (*Po0.01 vs mock+sh-Scb). (d) ChIP and real-time qPCR assays showing the binding of RNA Pol II, H3K4me2, H3K4me3, H3K9me3 and H3K27me3 to MYCN promoter in SH-SY5Y cells stably transfected with sh-Scb or sh-CTCF and co-transfected with mock or MYCNOS (*Po0.01 vs sh-Scb+mock).

Article Snippet: Immunohistochemistry Immunohistochemical staining and quantitative evaluation were performed as previously described,29–31 with antibodies specific for MYCN (sc22835) and CTCF (sc-271474, Santa Cruz Biotechnology; 1:200 dilutions).

Techniques: Binding Assay, Real-time Polymerase Chain Reaction, Stable Transfection, Transfection, shRNA, Plasmid Preparation

Journal: Oncogene

Article Title: CTCF cooperates with noncoding RNA MYCNOS to promote neuroblastoma progression through facilitating MYCN expression.

doi: 10.1038/onc.2015.422

Figure Lengend Snippet: Figure 6. CTCF and MYCNOS are highly expressed and positively correlated with MYCN expression in NB tissues and cell lines. (a) Immunohistochemical staining showing the expression of CTCF and MYCN in the tumor cells of NB specimens (arrowheads, brown). Scale bars: 100 μm. (b) Western blot assay indicating the protein levels of CTCF and MYCN in normal dorsal ganglia (DG), NB tissues (n = 3) and cultured cell lines (SH-SY5Y, SK-N-SH, IMR32 and BE(2)-C) (*Po0.01 vs DG). (c) Real-time quantitative RT–PCR assay revealing the transcript levels of CTCF, MYCNOS and MYCN in DG, NB tissues (n = 30) and cultured cell lines (SH-SY5Y, SK-N-SH, IMR32 and BE(2)-C) (*Po0.01 vs DG). (d, e) The positive correlation between MYCN and CTCF or MYCNOS transcript levels in NB tissues (n = 30). (f) Kaplan–Meier survival plots of 88 and 498 well-defined NB cases derived from R2 microarray analysis and visualization platform (http://r2.amc.nl).

Article Snippet: Immunohistochemistry Immunohistochemical staining and quantitative evaluation were performed as previously described,29–31 with antibodies specific for MYCN (sc22835) and CTCF (sc-271474, Santa Cruz Biotechnology; 1:200 dilutions).

Techniques: Expressing, Immunohistochemical staining, Staining, Western Blot, Cell Culture, Quantitative RT-PCR, Derivative Assay, Microarray

Journal: eLife

Article Title: Translation in amino-acid-poor environments is limited by tRNA Gln charging

doi: 10.7554/eLife.62307

Figure Lengend Snippet:

Article Snippet: The following primary antibodies were used: phospho-Thr899 GCN2 (ab75836, Abcam), total GCN2 (3302, Cell Signaling), phospho-Thr-389-S6K1 (9234, Cell Signaling), S6K1 (2708, Cell Signaling), vinculin (V9131, Sigma-Aldrich), ATF4 (sc-200, Santa Cruz), MED12 (A300-774A, Bethyl), TBP (A301-229A, Bethyl), CBP (A300-362A, Bethyl), CBFα1 (12556, Cell Signaling), BRD4 (ab128874, Abcam), CTCF (A700-041-T, Bethyl), RNA pol II (39497, Active Motif), Histone H3 (3638, Cell Signaling), α-Tubulin (T9026, Sigma-Aldrich), β-Actin (A5441, Sigma-Aldrich), HA tag (2367, Cell Signaling), puromycin (MABE343, EMD Millipore), and GLS (ab156876, Abcam).

Techniques: Recombinant, Control, shRNA, Luciferase, Plasmid Preparation, Expressing, Virus, Sequencing

Journal: PLoS ONE

Article Title: Construction of ceRNA network to identify the lncRNA and mRNA related to non-small cell lung cancer

doi: 10.1371/journal.pone.0259091

Figure Lengend Snippet: The primers used in this study.

Article Snippet: The membranes were incubated with the primary antibody CTCFL (60079-1-Ig, 1: 1000, proteintech), KRT5 (66727-1-Ig, 1: 10000, proteintech), LY6D (17361-1-AP, 1: 300, proteintech), TMEM (20995-1-AP, 1: 500, proteintech), GBP6 (23001-1-AP, 1: 1000, proteintech), TMEM179 (24799-1-AP, 1: 600, proteintech), β-actin (60008-1-Ig, 1: 5000, proteintech) overnight at 4°C.

Techniques:

Journal: PLoS ONE

Article Title: Construction of ceRNA network to identify the lncRNA and mRNA related to non-small cell lung cancer

doi: 10.1371/journal.pone.0259091

Figure Lengend Snippet: A. Survival curve of CTCFL. B. Survival curve of GBP6. C. Survival curve of KRT5. D. Survival curve of LY6D. E. Survival curve of TMEM. F. Survival curve of TMEM179. The abscissa represented survival time (days), and the ordinate represented survival rate. The blue line represented the low risk curve and the red line represents the high risk curve, p<0.05.

Article Snippet: The membranes were incubated with the primary antibody CTCFL (60079-1-Ig, 1: 1000, proteintech), KRT5 (66727-1-Ig, 1: 10000, proteintech), LY6D (17361-1-AP, 1: 300, proteintech), TMEM (20995-1-AP, 1: 500, proteintech), GBP6 (23001-1-AP, 1: 1000, proteintech), TMEM179 (24799-1-AP, 1: 600, proteintech), β-actin (60008-1-Ig, 1: 5000, proteintech) overnight at 4°C.

Techniques:

Journal: PLoS ONE

Article Title: Construction of ceRNA network to identify the lncRNA and mRNA related to non-small cell lung cancer

doi: 10.1371/journal.pone.0259091

Figure Lengend Snippet: qRT-PCR (A) and WB (B) were used to detect the expression levels of CTCFL, KRT5, LY6D, TMEM, GBP6, TMEM179. (C) Pearson analysis was used to detect the correlation between LINC00968 and CTCFL, GBP6, KRT5.

Article Snippet: The membranes were incubated with the primary antibody CTCFL (60079-1-Ig, 1: 1000, proteintech), KRT5 (66727-1-Ig, 1: 10000, proteintech), LY6D (17361-1-AP, 1: 300, proteintech), TMEM (20995-1-AP, 1: 500, proteintech), GBP6 (23001-1-AP, 1: 1000, proteintech), TMEM179 (24799-1-AP, 1: 600, proteintech), β-actin (60008-1-Ig, 1: 5000, proteintech) overnight at 4°C.

Techniques: Quantitative RT-PCR, Expressing

Journal: PLoS ONE

Article Title: Construction of ceRNA network to identify the lncRNA and mRNA related to non-small cell lung cancer

doi: 10.1371/journal.pone.0259091

Figure Lengend Snippet: (A) qRT-PCR was explored to examine whether knockdown of LINC00968 is successful. (B) CCK8 was performed to measure cell proliferation. qRT-PCR (C) and WB (D) were used to detect the expression of CTCFL, GBP6, KRT5. (E) Flow cytometry was utilized to detect cell apoptosis. (F) Tunel was explored to determined cell apoptosis.

Article Snippet: The membranes were incubated with the primary antibody CTCFL (60079-1-Ig, 1: 1000, proteintech), KRT5 (66727-1-Ig, 1: 10000, proteintech), LY6D (17361-1-AP, 1: 300, proteintech), TMEM (20995-1-AP, 1: 500, proteintech), GBP6 (23001-1-AP, 1: 1000, proteintech), TMEM179 (24799-1-AP, 1: 600, proteintech), β-actin (60008-1-Ig, 1: 5000, proteintech) overnight at 4°C.

Techniques: Quantitative RT-PCR, Knockdown, Expressing, Flow Cytometry, TUNEL Assay