Journal: Theranostics

Article Title: c-Myc is Required for BRAF V600E -Induced Epigenetic Silencing by H3K27me3 in Tumorigenesis

doi: 10.7150/thno.19884

Figure Lengend Snippet: Braf V600E signaling induces epigenetic silencing by increasing H3K27me3 abundance in NIH3T3 cells . ( A ) Western blot analysis of B-raf, phosphorylated (p-) and total forms of Erk1/2 and H3K27me3. GAPDH and histone H3 were used as loading controls. ( B ) Flow chart shows the experimental design. ( C ) Pie chart shows distribution of H3K27me3 enrichment across whole genome in Braf V600E cells relative to Braf WT cells. ( D ) Venn diagram illustrates that 150 loci were marked by H3K27me3 and downregulated by Braf V600E signaling.

Article Snippet: The rabbit polyclonal antibodies against H3K27me3 (#9733, CST), H3K4me3 (#9727, CST) and c-Myc (sc-764, Santa Cruz) were used for the ChIP assay using the Pierce™ Magnetic ChIP Kit (Thermo, Inc., MA, USA) according to the instructions of manufacturer.

Techniques: Western Blot

Journal: Theranostics

Article Title: c-Myc is Required for BRAF V600E -Induced Epigenetic Silencing by H3K27me3 in Tumorigenesis

doi: 10.7150/thno.19884

Figure Lengend Snippet: Validation of mRNA expression and H3K27me3 of Braf V600E -repressed genes in vitro and in vivo . ( A ) qRT-PCR analysis of Abi3bp , Gimap6 , Hopx , Pdzd2 , Ptprd , Rspo2 , Mtss1 and Nrxn3 expression in Braf V600E cells relative to that in Braf WT cells. 18S rRNA was used as an endogenous control. Data were presented as mean ± SD from three independent experiments. ( B ) ChIP-qPCR analysis of promoter regions of the indicated genes in Braf V600E cells relative to that in Braf WT cells using anti-H3K27me3 antibody. Data were presented as mean ± SD from three independent assays. ( C ) Comparison of the levels of p-Erk1/2 and H3K27me3 between TPO-Cre/LSL-Braf V600E (Braf V600E ) and Braf wild-type mice by IHC analysis. The inserts show the magnified image of the area indicated by the red square. Scale bars, 200 μm. ( D ) qRT-PCR analysis of Abi3bp , Gimap6 , Hopx , Pdzd2 , Ptprd , Rspo2 , Mtss1 and Nrxn3 expression in Braf V600E mice relative to that in Braf wild-type mice. 18S rRNA was used as an endogenous control. Data were presented as mean ± SD. Statistically significant differences were indicated: *, P <0.05; **, P <0.01.

Article Snippet: The rabbit polyclonal antibodies against H3K27me3 (#9733, CST), H3K4me3 (#9727, CST) and c-Myc (sc-764, Santa Cruz) were used for the ChIP assay using the Pierce™ Magnetic ChIP Kit (Thermo, Inc., MA, USA) according to the instructions of manufacturer.

Techniques: Expressing, In Vitro, In Vivo, Quantitative RT-PCR

Journal: Theranostics

Article Title: c-Myc is Required for BRAF V600E -Induced Epigenetic Silencing by H3K27me3 in Tumorigenesis

doi: 10.7150/thno.19884

Figure Lengend Snippet: The effect of inhibition of Braf V600E signaling on H3K27me3 levels and the expression of Braf V600E -repressed genes in Braf V600E mice. Comparison of tumor volume (A), weight (B), and the levels of p-Erk1/2 and H3K27me3 (C) between Braf V600E mice treated with a combination of PLX4720 and GSK1120212 (n=3) and vehicle (n=5), respectively. Scale bars, 200 μm. (D) qRT-PCR analysis of Abi3bp , Gimap6 , Hopx , Pdzd2 , Ptprd , Rspo2 , Mtss1 and Nrxn3 expression in combination-treated mice relative to that in vehicle-treated mice. DMSO was used as the vehicle control. 18S rRNA was used as an endogenous control. Data were presented as mean ± SD. Statistically significant differences were indicated: *, P <0.05; **, P <0.01; ***, P <0.001.

Article Snippet: The rabbit polyclonal antibodies against H3K27me3 (#9733, CST), H3K4me3 (#9727, CST) and c-Myc (sc-764, Santa Cruz) were used for the ChIP assay using the Pierce™ Magnetic ChIP Kit (Thermo, Inc., MA, USA) according to the instructions of manufacturer.

Techniques: Inhibition, Expressing, Quantitative RT-PCR

Journal: Theranostics

Article Title: c-Myc is Required for BRAF V600E -Induced Epigenetic Silencing by H3K27me3 in Tumorigenesis

doi: 10.7150/thno.19884

Figure Lengend Snippet: Braf V600E increases H3K27me3 levels through regulating the PRC2 components via c-Myc . ( A ) Western blot analysis of B-raf, p-Erk1/2, total Erk1/2, c-Myc, Ezh2, Suz12, Jarid2 and H3K27me3 in VEC, Braf WT and Braf V600E cells, respectively. β-Actin and histone H3 were used as loading controls. ( B ) Western blot analysis of Ezh2, Suz12, Jarid2 and H3K27me3 upon c-Myc depletion in NIH3T3 cells. β-Actin and histone H3 were used as loading controls. si-NC, si-Myc1 and 2 represent control and specific c-Myc siRNAs, respectively. ( C ) Western blot analysis of Ezh2, Suz12, Jarid2 and H3K27me3 in Braf V600E cells relative to that in Braf WT cells. β-Actin and histone H3 were used as loading controls. ( D ) A comparison of the levels of c-Myc, Ezh2, Suz12, Jarid2 and H3K27me3 in the indicated mice by IHC staining. DMSO was used as the vehicle control. The insert shows the magnified image of the area indicated by the red square. Scale bars, 200 μm.

Article Snippet: The rabbit polyclonal antibodies against H3K27me3 (#9733, CST), H3K4me3 (#9727, CST) and c-Myc (sc-764, Santa Cruz) were used for the ChIP assay using the Pierce™ Magnetic ChIP Kit (Thermo, Inc., MA, USA) according to the instructions of manufacturer.

Techniques: Western Blot, Immunohistochemistry

Journal: Theranostics

Article Title: c-Myc is Required for BRAF V600E -Induced Epigenetic Silencing by H3K27me3 in Tumorigenesis

doi: 10.7150/thno.19884

Figure Lengend Snippet: The effect of inhibition of c-Myc by 10058-F4 on the levels of the PRC2 components and H3K27me3 in Braf V600E mice . Comparison of tumor volume ( A ), weight ( B ), and the levels of Ncl, Ezh2, Suz12, Jarid2 and H3K27me3 (C) between Braf V600E mice treated with 10058-F4 (n=3) and vehicle (n=3), respectively. DMSO was used as the vehicle control. The insert shows the magnified image of the area indicated by the red square. Scale bars, 200 μm. ( D ) qRT-PCR analysis of Abi3bp , Gimap6 , Hopx , Pdzd2 , Ptprd , Rspo2 , Mtss1 and Nrxn3 expression in 10058-F4-treated mice relative to that in vehicle-treated mice. DMSO was used as the vehicle control. 18S rRNA was used as an endogenous control. Data were presented as mean ± SD. Statistically significant differences were indicated: *, P <0.05; **, P <0.01; ***, P <0.001.

Article Snippet: The rabbit polyclonal antibodies against H3K27me3 (#9733, CST), H3K4me3 (#9727, CST) and c-Myc (sc-764, Santa Cruz) were used for the ChIP assay using the Pierce™ Magnetic ChIP Kit (Thermo, Inc., MA, USA) according to the instructions of manufacturer.

Techniques: Inhibition, Quantitative RT-PCR, Expressing

Journal: Theranostics

Article Title: c-Myc is Required for BRAF V600E -Induced Epigenetic Silencing by H3K27me3 in Tumorigenesis

doi: 10.7150/thno.19884

Figure Lengend Snippet: The effect of inhibition of major signaling pathways on the levels of the PRC2 components and H3K27me3 in thyroid cancer and melanoma cells. Western blot was performed to evaluate the effect of the indicated inhibitors individually or in combinations on the levels of the PRC2 components and H3K27me3 in thyroid cancer cell lines BCPAP and K1 (A) and melanoma cell lines M14 and A375 (B). GAPDH and histone H3 were used as loading controls. DMSO was used as the vehicle control.

Article Snippet: The rabbit polyclonal antibodies against H3K27me3 (#9733, CST), H3K4me3 (#9727, CST) and c-Myc (sc-764, Santa Cruz) were used for the ChIP assay using the Pierce™ Magnetic ChIP Kit (Thermo, Inc., MA, USA) according to the instructions of manufacturer.

Techniques: Inhibition, Western Blot

Journal: Theranostics

Article Title: c-Myc is Required for BRAF V600E -Induced Epigenetic Silencing by H3K27me3 in Tumorigenesis

doi: 10.7150/thno.19884

Figure Lengend Snippet: Schematic model of BRAF V600E signaling-induced epigenetic silencing by H3K27me3 in tumorigenesis . BRAF V600E mutation constitutively activates the MAPK/Erk signaling, resulting in upregulation of its downstream effector c-Myc in many types of cancer particularly thyroid cancer and melanoma. In general, c-Myc is degraded via the ubiquitin-proteasome system. However, this process can be inhibited by activated PI3K/Akt pathway in cancer cells. When c-Myc translocates into the nucleus, it regulates the expression of its target genes through binding to their regulatory elements via interaction with its obligate partner MAX or other unknown mechanisms. In the present study, c-Myc regulates the expression of the PRC2 components, contributing to epigenetic silencing through two distinct mechanisms. First, c-Myc transcriptionally regulates the expression of the PRC2 components through binding to the regulatory elements within their promoters. Second, c-Myc transcriptionally represses the expression of a panel of miRNAs, whereas some of them target and regulate the expression of the PRC2 components at post-transcriptional levels. In addition, BRAF V600E signaling also induces epigenetic silencing through Erk1/2-induced RNAPII poising and chromatin architecture.

Article Snippet: The rabbit polyclonal antibodies against H3K27me3 (#9733, CST), H3K4me3 (#9727, CST) and c-Myc (sc-764, Santa Cruz) were used for the ChIP assay using the Pierce™ Magnetic ChIP Kit (Thermo, Inc., MA, USA) according to the instructions of manufacturer.

Techniques: Mutagenesis, Expressing, Binding Assay

Journal: PLoS ONE

Article Title: The Nucleosome (Histone-DNA Complex) Is the TLR9-Specific Immunostimulatory Component of Plasmodium falciparum That Activates DCs

doi: 10.1371/journal.pone.0020398

Figure Lengend Snippet: Panel A : Fractionation of MZs lysate by sucrose-density gradient centrifugation. The values of absorption at 260 nm (ℓ) and 280 nm ( ), and DNA contents (O) of the fractions were measured as described in “ ”, and plotted. Panel B : SDS-PAGE analysis. The sucrose gradient fractions were dialyzed; aliquots corresponding to 20 µg of proteins were analyzed by SDS-PAGE using 15% gels. Sucrose gradient fraction numbers are indicated at the top. The mobility of molecular weight marker proteins is indicated to the left. Lane S, a mixture of standard H1, H2A, H2B, H3 and H4 histones. Panel C : Western blotting of sucrose gradient fractions using anti-H3 histone polyclonal antibodies. Lane descriptions are same as that in panel B . Panels D and E : TNF-α and IL-12 produced by WT FL-DCs stimulated with the indicated sucrose gradient fractions having 2.5 µg/ml of DNA content or with fractions having 2.5 µg/ml of DNA content to which the purified parasite genomic DNA (pDNA, 8 µg/ml) was added. Cells stimulated with parasite genomic DNA were used as controls. Panels F and G : TNF-α and IL-12 produced by FL-DCs from WT, TLR2 −/− , TLR9 −/− and MyD88 −/− stimulated with sucrose gradient fractions. DCs stimulated with Pam 3 CSK 4 , Poly I∶C, LPS, CpG ODN or parasite genomic DNA were used as a control. The analysis was repeated three times each time performed in duplicates, and results of a representative are shown. Error bars represent mean values ± SEM. * p <0.05; ** p <0.01 comparison between sucrose gradient fractions with and without added parasite DNA.

Article Snippet: Rabbit anti-human H3 histone polyclonal antibodies was from Cell Signaling Technology (Beverly, MA).

Techniques: Fractionation, Gradient Centrifugation, SDS Page, Molecular Weight, Marker, Western Blot, Produced, Purification

Journal: PLoS ONE

Article Title: The Nucleosome (Histone-DNA Complex) Is the TLR9-Specific Immunostimulatory Component of Plasmodium falciparum That Activates DCs

doi: 10.1371/journal.pone.0020398

Figure Lengend Snippet: Panel A : The parasite nuclear material, polynucleosomes and buffer/salt extracts of nuclear material were analyzed for proteins by SDS-PAGE (in each lane 15 µg of protein content/well). Lane 1, the supernatant of parasite lysate (see for materials in lanes 1–6); lane 2, 0.3 M KCl extract of the nuclear material; lane 3, 0.6 M KCl extract of the chromatin material; lane 4, 0.4 M NaCl extract of the chromatin material; lane 5, chromatin material after extraction of proteins with buffer A containing various salts; lane 6, polynucleosomes; lane 7, the mixture of standard recombinant histones, H1, H2A, H2B, H3 and H4. The mobility of molecular weight marker proteins is indicated to the right. Panel B : The parasite nuclear material and buffer/salt extracts of nuclear material were electrophoresed on 0.8% agarose gels and the DNA bands were visualized under UV light after ethidium bromide staining. Lane 1, parasite nuclear material before extraction of proteins that loosely bound to chromatin (see for materials in lanes 1–10); lane 2, chromatin material pellet after extraction with buffer/0.3 M KCl; lane 3, chromatin material after extraction with buffer/0.6 M KCl; lane 4, chromatin material after extraction with buffer/0.4 M NaCl; lane 5, soluble polynucleosomes; lane 6, insoluble fibrous material after obtaining polynucleosomes; lane 7, parasite cytoplasmic material plus membrane fragments; lane 8, 0.3 M KCl extract of nuclear material; lane 9, 0.6 M KCl extract; lane 10, 0.4 M NaCl extract. Lanes 1–5, materials having 0.5 µg DNA were analyzed. Lane 6, about 60-fold more material as compared to those in lanes 1–5 based on the total parasite amount used for fractionation. Lanes 7–10, materials equivalent to that in lanes 1–5 based on the total amount of parasite used for fractionation was loaded. The sizes of standard DNA makers are indicated to the right. Panel C : Western blotting of parasite components as shown in panel A was done using anti-H3 histone polyclonal antibodies. Lane descriptions are as outlined in panel A .

Article Snippet: Rabbit anti-human H3 histone polyclonal antibodies was from Cell Signaling Technology (Beverly, MA).

Techniques: SDS Page, Recombinant, Molecular Weight, Marker, Staining, Fractionation, Western Blot

Journal: PLoS ONE

Article Title: The Human Gonadotropin Releasing Hormone Type I Receptor Is a Functional Intracellular GPCR Expressed on the Nuclear Membrane

doi: 10.1371/journal.pone.0011489

Figure Lengend Snippet: (A) Purity assessment of nuclei preparations used in functional assays by bright field microscopy and Western blotting. Left panel: Photomicrograph of a typical nuclei preparation following trypan blue staining. Scale bar = 10 µm. Insert: digital magnification of a single nucleus showing the presence of nucleoles (arrow). Right panel: Immunodetection of the membrane and nuclear markers clathrin and lamin A/C respectively in membrane and isolated nuclei fractions from FLAG-GnRH-R-transfected HEK 293 cells. (B) Time course of Histone H3-Lys9/Lys14 acetylation and -Ser10 phosphorylation in GnRH-stimulated nuclei by Western blot analysis. A representative Western blot is shown. Densitometric values of acetylated and phosphorylated Histone H3 levels were normalized to those of total Histone H3 and the relative intensity ratios were expressed in-fold increase over time zero. Data are means ± S.E.M of four independent experiments. * P <0.05 vs control time zero. Statistical analyses performed using one-way ANOVA followed by Dunnet's ad hoc test.

Article Snippet: Fetal bovine serum (FBS), collagen, rabbit anti-FLAG antibody and Buserelin were purchased from Sigma Aldrich Inc. (Oakville, ON, Canada), mouse anti-lamin A/C, and mouse anti-calnexin from AbCam (Cambridge, MA, USA), mouse anti-GM130 from Transduction Laboratories (BD Biosciences, Mississauga, ON, Canada), murine anti-clathrin monoclonal antibody from Santa-Cruz Biotechnology (Santa-Cruz, CA, USA), rabbit anti-Histone H3 and rabbit anti-phospho-Histone H3 (Ser10) polyclonal antibodies from Cell Signaling (Danvers, MA, USA) and rabbit anti-acetyl-Histone H3 polyclonal (Lys9 and Lys14) antibody from Upstate Biotechnology (Lake Placid, NY, USA).

Techniques: Functional Assay, Microscopy, Western Blot, Staining, Immunodetection, Isolation, Transfection