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p38 SAPK recruitment at the c-Fos promoter requires the transcription factor <t>Elk1.</t> A , HeLa cells were transfected with either control or Elk1 <t>siRNAs</t> and the cell lysates were inmunoprecipitated with anti-Elk1 antibody. Both inputs and immunoprecipitates were analyzed by Western blotting with anti-Elk1 antibody. * marks a nonspecific band. B , HeLa cells were transfected with the indicated siRNAs and treated with 100 m m NaCl for 2 h. The mRNA levels of c-Fos, Elk1, and GAPDH were analyzed by PCR. C , HeLa cells were treated with 100 m m NaCl for 60 min either in the presence or absence SB203580 and subjected to ChIP assay using an anti-Elk1 antibody. D , HeLa cells were transfected with pCDNA3–3HA-p38α along with control or Elk1 siRNAs. Cells were treated with 100 m m NaCl for 60 min and subjected to ChIP assay with anti-HA antibody. Immunoprecipitated DNA fragments were subjected to PCR analysis.
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1) Product Images from "The p38 SAPK Is Recruited to Chromatin via Its Interaction with Transcription Factors *"

Article Title: The p38 SAPK Is Recruited to Chromatin via Its Interaction with Transcription Factors *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.155846

p38 SAPK recruitment at the c-Fos promoter requires the transcription factor Elk1. A , HeLa cells were transfected with either control or Elk1 siRNAs and the cell lysates were inmunoprecipitated with anti-Elk1 antibody. Both inputs and immunoprecipitates were analyzed by Western blotting with anti-Elk1 antibody. * marks a nonspecific band. B , HeLa cells were transfected with the indicated siRNAs and treated with 100 m m NaCl for 2 h. The mRNA levels of c-Fos, Elk1, and GAPDH were analyzed by PCR. C , HeLa cells were treated with 100 m m NaCl for 60 min either in the presence or absence SB203580 and subjected to ChIP assay using an anti-Elk1 antibody. D , HeLa cells were transfected with pCDNA3–3HA-p38α along with control or Elk1 siRNAs. Cells were treated with 100 m m NaCl for 60 min and subjected to ChIP assay with anti-HA antibody. Immunoprecipitated DNA fragments were subjected to PCR analysis.
Figure Legend Snippet: p38 SAPK recruitment at the c-Fos promoter requires the transcription factor Elk1. A , HeLa cells were transfected with either control or Elk1 siRNAs and the cell lysates were inmunoprecipitated with anti-Elk1 antibody. Both inputs and immunoprecipitates were analyzed by Western blotting with anti-Elk1 antibody. * marks a nonspecific band. B , HeLa cells were transfected with the indicated siRNAs and treated with 100 m m NaCl for 2 h. The mRNA levels of c-Fos, Elk1, and GAPDH were analyzed by PCR. C , HeLa cells were treated with 100 m m NaCl for 60 min either in the presence or absence SB203580 and subjected to ChIP assay using an anti-Elk1 antibody. D , HeLa cells were transfected with pCDNA3–3HA-p38α along with control or Elk1 siRNAs. Cells were treated with 100 m m NaCl for 60 min and subjected to ChIP assay with anti-HA antibody. Immunoprecipitated DNA fragments were subjected to PCR analysis.

Techniques Used: Transfection, Western Blot, Polymerase Chain Reaction, Chromatin Immunoprecipitation, Immunoprecipitation

2) Product Images from "Dynamic recruitment of transcription factors and epigenetic changes on the ER stress response gene promoters"

Article Title: Dynamic recruitment of transcription factors and epigenetic changes on the ER stress response gene promoters

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkl304

Binding of NF-Y, Sp1 and p53 to ER stress promoters. ChIP analysis of NF-Y, Sp1 and p53 in HepG2 cells. Chromatin was prepared from cells treated for the indicated times with Tg. Two semi-quantitative PCR amplifications are shown for most targets. The number of PCR cycles are shown on the left.
Figure Legend Snippet: Binding of NF-Y, Sp1 and p53 to ER stress promoters. ChIP analysis of NF-Y, Sp1 and p53 in HepG2 cells. Chromatin was prepared from cells treated for the indicated times with Tg. Two semi-quantitative PCR amplifications are shown for most targets. The number of PCR cycles are shown on the left.

Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

3) Product Images from "Wnt/?-Catenin Signaling Enhances Cyclooxygenase-2 (COX2) Transcriptional Activity in Gastric Cancer Cells"

Article Title: Wnt/?-Catenin Signaling Enhances Cyclooxygenase-2 (COX2) Transcriptional Activity in Gastric Cancer Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0018562

Binding of β-catenin to the TBE Site II (−689/−684) in the COX2 promoter. (A–C) ChIP assays in MKN45 cells using specific antibodies for β-catenin (β-cat), polymerase II (Pol), H3 and H4 acetylated histones (H3ac; H4ac) and immunoglobulin G (IgG). Quantification was done by real time PCR using specific primers for the proximal promoter (PP) region (A), the TBE Site II (−689/−684) in the human COX2 promoter (B) and a TBE site within the c-myc promoter, as a positive control (C). (D) EMSA assay in nuclear extracts from MKN45 cells. DNA-protein complexes formed by incubating nuclear extracts (N.E.) from MKN45 cells with radiolabelled probes containing the intact and a mutated TBE Site II (lanes 3 and 5) were resolved in native polyacrylamide gels at 5% and revealed through autoradiography. To determine the specificity of the binding a 50 times excess of non-radiolabelled wild-type (line 4) and mutant (6 and 7) oligonucleotides were added. Lanes 1 and 2 correspond to the wild-type and mutant radiolabelled oligonucleotides without incubation with N.E. These tests are representative of three independent experiments. E. Model depicting the molecular mechanism by which Wnt/β-catenin signaling may contribute to the expression of the human COX2 gene.
Figure Legend Snippet: Binding of β-catenin to the TBE Site II (−689/−684) in the COX2 promoter. (A–C) ChIP assays in MKN45 cells using specific antibodies for β-catenin (β-cat), polymerase II (Pol), H3 and H4 acetylated histones (H3ac; H4ac) and immunoglobulin G (IgG). Quantification was done by real time PCR using specific primers for the proximal promoter (PP) region (A), the TBE Site II (−689/−684) in the human COX2 promoter (B) and a TBE site within the c-myc promoter, as a positive control (C). (D) EMSA assay in nuclear extracts from MKN45 cells. DNA-protein complexes formed by incubating nuclear extracts (N.E.) from MKN45 cells with radiolabelled probes containing the intact and a mutated TBE Site II (lanes 3 and 5) were resolved in native polyacrylamide gels at 5% and revealed through autoradiography. To determine the specificity of the binding a 50 times excess of non-radiolabelled wild-type (line 4) and mutant (6 and 7) oligonucleotides were added. Lanes 1 and 2 correspond to the wild-type and mutant radiolabelled oligonucleotides without incubation with N.E. These tests are representative of three independent experiments. E. Model depicting the molecular mechanism by which Wnt/β-catenin signaling may contribute to the expression of the human COX2 gene.

Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Positive Control, Autoradiography, Mutagenesis, Incubation, Expressing

4) Product Images from "miR675 Accelerates Malignant Transformation of Mesenchymal Stem Cells by Blocking DNA Mismatch Repair"

Article Title: miR675 Accelerates Malignant Transformation of Mesenchymal Stem Cells by Blocking DNA Mismatch Repair

Journal: Molecular Therapy. Nucleic Acids

doi: 10.1016/j.omtn.2018.11.010

miR675 Triggers MSI and Abnormal Gene Expression in the Human Mesenchymal Stem Cells Infected with rLV and rLV-miR675, Respectively (A) Microsatellite instability (MSI) analysis through dot blot (slot blot) using various biotin-labeling MSI probes (biotin-MSIs). (B) (a) Chromosome conformation capture (3C)-chromatin immunoprecipitation (ChIP) with anti-P300 and anti-Pol II. The chromatin was cross-linked, digested with restriction enzymes, and ligated under conditions that favor intramolecular ligation. Immediately after ligation, the chromatin was immunoprecipitated using an antibody (anti-P300, anti-Pol II) against the protein of interest. Thereafter, the cross-links were reversed and the DNA was purified further. The PCR anlysis was applied for detecting CyclinD1 promoter-enhancer coupling product using CyclinD1 promoter and enhancer primers. The CyclinD1 promoter and enhancer was the INPUT. (b) The quantitative analysis of ChIP-3C. (C) (a) Western blotting with anti-Rad51, anti-CDK2, anti-CyclinE, anti-CDK4, anti-CyclinD1, anti-PCNA, anti-ppRB, anti-E2F1, anti-P18, anti-P21, anti-PKM2, anti-c-Myc, and anti-Chk1. β-actin was the internal control. (b) The gray scan analysis of positive bands of western blotting.
Figure Legend Snippet: miR675 Triggers MSI and Abnormal Gene Expression in the Human Mesenchymal Stem Cells Infected with rLV and rLV-miR675, Respectively (A) Microsatellite instability (MSI) analysis through dot blot (slot blot) using various biotin-labeling MSI probes (biotin-MSIs). (B) (a) Chromosome conformation capture (3C)-chromatin immunoprecipitation (ChIP) with anti-P300 and anti-Pol II. The chromatin was cross-linked, digested with restriction enzymes, and ligated under conditions that favor intramolecular ligation. Immediately after ligation, the chromatin was immunoprecipitated using an antibody (anti-P300, anti-Pol II) against the protein of interest. Thereafter, the cross-links were reversed and the DNA was purified further. The PCR anlysis was applied for detecting CyclinD1 promoter-enhancer coupling product using CyclinD1 promoter and enhancer primers. The CyclinD1 promoter and enhancer was the INPUT. (b) The quantitative analysis of ChIP-3C. (C) (a) Western blotting with anti-Rad51, anti-CDK2, anti-CyclinE, anti-CDK4, anti-CyclinD1, anti-PCNA, anti-ppRB, anti-E2F1, anti-P18, anti-P21, anti-PKM2, anti-c-Myc, and anti-Chk1. β-actin was the internal control. (b) The gray scan analysis of positive bands of western blotting.

Techniques Used: Expressing, Infection, Dot Blot, Labeling, Chromatin Immunoprecipitation, Ligation, Immunoprecipitation, Purification, Polymerase Chain Reaction, Western Blot

5) Product Images from "Smad4-dependent desmoglein-4 expression contributes to hair follicle integrity"

Article Title: Smad4-dependent desmoglein-4 expression contributes to hair follicle integrity

Journal: Developmental biology

doi: 10.1016/j.ydbio.2008.07.020

Keratinocyte-specific Smad4 deletion A: Schematic demonstrating generation of K5.Smad4−/− mice by mating K5.CrePR1 mice with Smad4 floxed mice (Smad4f/f), and applying RU486 to bigenic mouse skin. B: Genomic PCR results from E10.5 mouse tail DNA indicate genotype of Smad4 alleles. Lanes 2 and 3 = K5.CrePR1/Smad4f/wt and K5.CrePR1/Smad4f/f, which were heterozygous and homozygous for the floxed allele (FL), respectively and both were positive for the CrePR1 transgene (cre). Smad4 deletion (ΔSmad4) was induced by RU486 application to day 9.5 pregnant bigenic mice. C to I: Immunostaining for Smad4 (brown) of skins of E12.5 (C and D), E15.5 (E and F), and P35 (G to I). Note that Smad4 nuclear staining was uniform in WT E12.5 epidermis, but was patchy and reduced in K5.Smad4−/− E12.5 epidermis. Smad4 nuclear positive cells were predominantly located in the basal layer of the epidermis and the hair follicle placode (arrow), and stromal cells in WT E15.5 skin. Smad4 protein was ablated in the epidermis and placode (arrow) in K5.Smad4 −/− skin, but was still detected stromal cells. In P35 WT skin, Smad4 staining was positive in all layers of the anagen follicle (G), the epidermis (H), and some stromal cells (G and H). In contrast, in P35 K5.Smad4−/− skin, Smad4 staining was negative in the epidermis, degenerated hair follicle cysts and sebaceous glands (I). The stroma remained Smad4 positive cells. Light brown staining in K5.Smad4−/− epidermis, hair follicle cysts and sebaceous glands represents background staining. Scale bars = C–D: 25µm E–F: 50µm G–I: 100µm.)
Figure Legend Snippet: Keratinocyte-specific Smad4 deletion A: Schematic demonstrating generation of K5.Smad4−/− mice by mating K5.CrePR1 mice with Smad4 floxed mice (Smad4f/f), and applying RU486 to bigenic mouse skin. B: Genomic PCR results from E10.5 mouse tail DNA indicate genotype of Smad4 alleles. Lanes 2 and 3 = K5.CrePR1/Smad4f/wt and K5.CrePR1/Smad4f/f, which were heterozygous and homozygous for the floxed allele (FL), respectively and both were positive for the CrePR1 transgene (cre). Smad4 deletion (ΔSmad4) was induced by RU486 application to day 9.5 pregnant bigenic mice. C to I: Immunostaining for Smad4 (brown) of skins of E12.5 (C and D), E15.5 (E and F), and P35 (G to I). Note that Smad4 nuclear staining was uniform in WT E12.5 epidermis, but was patchy and reduced in K5.Smad4−/− E12.5 epidermis. Smad4 nuclear positive cells were predominantly located in the basal layer of the epidermis and the hair follicle placode (arrow), and stromal cells in WT E15.5 skin. Smad4 protein was ablated in the epidermis and placode (arrow) in K5.Smad4 −/− skin, but was still detected stromal cells. In P35 WT skin, Smad4 staining was positive in all layers of the anagen follicle (G), the epidermis (H), and some stromal cells (G and H). In contrast, in P35 K5.Smad4−/− skin, Smad4 staining was negative in the epidermis, degenerated hair follicle cysts and sebaceous glands (I). The stroma remained Smad4 positive cells. Light brown staining in K5.Smad4−/− epidermis, hair follicle cysts and sebaceous glands represents background staining. Scale bars = C–D: 25µm E–F: 50µm G–I: 100µm.)

Techniques Used: Mouse Assay, Polymerase Chain Reaction, Immunostaining, Staining

Early onset of hair follicle degeneration in K5.Smad4−/− skin A: A P6 K5.Smad4−/− mouse (−/−) was slightly smaller than its wildtype (WT) littermate. The arrow in C points to a degenerated hair follicle, which is enlarged in D. E: an example of a degenerated hair follicle from a P8 Dsg4 mutant (LahJ) skin for comparison. The “[“ symbols in D and E highlight the degenerated region of the hair follicle. F: A P10 K5.Smad4 −/− mouse (−/−) showed a smaller size than its wildtype (WT) littermate. G–I: Histology of P10 skins from WT (G), K5.Smad4−/− (H, −/−) and Dsg4 mutant (I, LahJ) mice. Arrows in H and I point to examples of degenerated hair follicles. Scale bars: B, C, G: 100µm; H, I: 150µm.
Figure Legend Snippet: Early onset of hair follicle degeneration in K5.Smad4−/− skin A: A P6 K5.Smad4−/− mouse (−/−) was slightly smaller than its wildtype (WT) littermate. The arrow in C points to a degenerated hair follicle, which is enlarged in D. E: an example of a degenerated hair follicle from a P8 Dsg4 mutant (LahJ) skin for comparison. The “[“ symbols in D and E highlight the degenerated region of the hair follicle. F: A P10 K5.Smad4 −/− mouse (−/−) showed a smaller size than its wildtype (WT) littermate. G–I: Histology of P10 skins from WT (G), K5.Smad4−/− (H, −/−) and Dsg4 mutant (I, LahJ) mice. Arrows in H and I point to examples of degenerated hair follicles. Scale bars: B, C, G: 100µm; H, I: 150µm.

Techniques Used: Mutagenesis, Mouse Assay

Loss of Smad4 results in progressive alopecia A: Anterior to posterior alopecia in P14 K5.Smad4−/− skin (−/−). When wildtype (WT) hair follicles were preparing to enter catagen (B), K5.Smad4 −/− (−/−) hair follicles (C) were shorter than WT follicles in B. K5.Smad4 −/− (−/−) hair follicles formed canals and lacked hair shafts, which was a more severe phenotype than Dsg4 mutant (LahJ) hair follicle degeneration shown in D. K5.Smad4−/− skin also exhibited prominent sebaceous glands, which was not shown in P14 Dsg4 mutant (LahJ) skin (D). E: Complete hairless of P35 K5.Smad4−/− mouse (−/−). F: Wildtype (WT) hair follicles entered into the second anagen growth phase while the Smad4 −/− hair follicles were degenerated (G). H: Adult Dsg4 mutant (LahJ) skin showing that all hair follicles were degenerated into cysts. Scale bars: B–D and F: 300µm. G and H:150 µm.
Figure Legend Snippet: Loss of Smad4 results in progressive alopecia A: Anterior to posterior alopecia in P14 K5.Smad4−/− skin (−/−). When wildtype (WT) hair follicles were preparing to enter catagen (B), K5.Smad4 −/− (−/−) hair follicles (C) were shorter than WT follicles in B. K5.Smad4 −/− (−/−) hair follicles formed canals and lacked hair shafts, which was a more severe phenotype than Dsg4 mutant (LahJ) hair follicle degeneration shown in D. K5.Smad4−/− skin also exhibited prominent sebaceous glands, which was not shown in P14 Dsg4 mutant (LahJ) skin (D). E: Complete hairless of P35 K5.Smad4−/− mouse (−/−). F: Wildtype (WT) hair follicles entered into the second anagen growth phase while the Smad4 −/− hair follicles were degenerated (G). H: Adult Dsg4 mutant (LahJ) skin showing that all hair follicles were degenerated into cysts. Scale bars: B–D and F: 300µm. G and H:150 µm.

Techniques Used: Mutagenesis

Loss of Dsg4 in K5.Smad4−/−hair follicles A–F: Immunofluorescence of Dsg4 (green) counterstained with K5 (red). Arrows in B and D point to the loss of Dsg4 protein in the precortex region of K5.Smad4−/− hair follicles. Scale bars: A, E=50µm B, C D = 100µm F = 200µm. G–H: qRT-PCR of dsg4 transcripts from K5.Smad4−/− dorsal skins (KO) compared to their wildtype (WT) littermate skins (3 skins/group). **: p
Figure Legend Snippet: Loss of Dsg4 in K5.Smad4−/−hair follicles A–F: Immunofluorescence of Dsg4 (green) counterstained with K5 (red). Arrows in B and D point to the loss of Dsg4 protein in the precortex region of K5.Smad4−/− hair follicles. Scale bars: A, E=50µm B, C D = 100µm F = 200µm. G–H: qRT-PCR of dsg4 transcripts from K5.Smad4−/− dorsal skins (KO) compared to their wildtype (WT) littermate skins (3 skins/group). **: p

Techniques Used: Immunofluorescence, Quantitative RT-PCR

Smad4 transcriptionally activates Dsg4 expression A: ChIP PCR shows that Smad1, 4 and 5, but not Smad2 and 3 bound to the promoter of Dsg4. B: PCR encompassing the Dsg4 promoter region without the SBE shows no Smad binding. Mouse (Ms) IgG and rabbit (Rbt) IgG were used as negative controls for Smad antibody. Antibody to RNA polymerase (pol) II was used as a positive control for ChIP assay. A Gata-3 antibody was used as a control for specificities of individual Smad antibodies. C: Positive control for BMP-specific Smad binding to the Msx2 promoter. D: PCR encompassing the Msx2 promoter region without the SBE shows no Smad binding. E: Luciferase reporter assay in K5.Smad4−/− keratinocytes for Dsg4 transcription with individual Smad (S) expression vectors. *: p
Figure Legend Snippet: Smad4 transcriptionally activates Dsg4 expression A: ChIP PCR shows that Smad1, 4 and 5, but not Smad2 and 3 bound to the promoter of Dsg4. B: PCR encompassing the Dsg4 promoter region without the SBE shows no Smad binding. Mouse (Ms) IgG and rabbit (Rbt) IgG were used as negative controls for Smad antibody. Antibody to RNA polymerase (pol) II was used as a positive control for ChIP assay. A Gata-3 antibody was used as a control for specificities of individual Smad antibodies. C: Positive control for BMP-specific Smad binding to the Msx2 promoter. D: PCR encompassing the Msx2 promoter region without the SBE shows no Smad binding. E: Luciferase reporter assay in K5.Smad4−/− keratinocytes for Dsg4 transcription with individual Smad (S) expression vectors. *: p

Techniques Used: Expressing, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Binding Assay, Mass Spectrometry, Positive Control, Luciferase, Reporter Assay

6) Product Images from "TCF7L1 Modulates Colorectal Cancer Growth by Inhibiting Expression of the Tumor-Suppressor Gene EPHB3"

Article Title: TCF7L1 Modulates Colorectal Cancer Growth by Inhibiting Expression of the Tumor-Suppressor Gene EPHB3

Journal: Scientific Reports

doi: 10.1038/srep28299

Knockdown of EPHB3 rescues growth of TCF7L1-Null cells. ( A ) Immunohistochemistry for EPHB3 shows elevated expression in TCF7L1-Null xenograft tumors (bottom) compared to control tumors from the same mice (top), reflecting the significant mRNA upregulation observed by RNA-sequencing and qPCR. ( B ) EPHB3 knockdown largely rescued colony formation of TCF7L1-Null cells. Colony number and average colony size was significantly higher than that of TCF7L1-Null cells, and almost returned to the levels of control cells (*P
Figure Legend Snippet: Knockdown of EPHB3 rescues growth of TCF7L1-Null cells. ( A ) Immunohistochemistry for EPHB3 shows elevated expression in TCF7L1-Null xenograft tumors (bottom) compared to control tumors from the same mice (top), reflecting the significant mRNA upregulation observed by RNA-sequencing and qPCR. ( B ) EPHB3 knockdown largely rescued colony formation of TCF7L1-Null cells. Colony number and average colony size was significantly higher than that of TCF7L1-Null cells, and almost returned to the levels of control cells (*P

Techniques Used: Immunohistochemistry, Expressing, Mouse Assay, RNA Sequencing Assay, Real-time Polymerase Chain Reaction

7) Product Images from "Human Beta Defensin 2 Selectively Inhibits HIV-1 in Highly Permissive CCR6+CD4+ T Cells"

Article Title: Human Beta Defensin 2 Selectively Inhibits HIV-1 in Highly Permissive CCR6+CD4+ T Cells

Journal: Viruses

doi: 10.3390/v9050111

hBD2 enhances binding of NFAT and IRF-4 to the APOBEC3G promoter. JKT-FT7 CCR6 GFP cells were treated with hBD2 (20 μg/mL) for the indicated timepoints. Chromatin immunoprecipitation (ChIP) assays were performed using antibodies against NFATc2, NFATc1, or IRF-4. Immunoprecipitated DNA was detected by quantitative real-time PCR performed in triplicate. Shown is fold enrichment determined by comparison with IgG control of three independent experiments. Error bars representing SEM were included. Statistical analysis was performed by paired 2-tail Student t test. * p
Figure Legend Snippet: hBD2 enhances binding of NFAT and IRF-4 to the APOBEC3G promoter. JKT-FT7 CCR6 GFP cells were treated with hBD2 (20 μg/mL) for the indicated timepoints. Chromatin immunoprecipitation (ChIP) assays were performed using antibodies against NFATc2, NFATc1, or IRF-4. Immunoprecipitated DNA was detected by quantitative real-time PCR performed in triplicate. Shown is fold enrichment determined by comparison with IgG control of three independent experiments. Error bars representing SEM were included. Statistical analysis was performed by paired 2-tail Student t test. * p

Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Immunoprecipitation, Real-time Polymerase Chain Reaction

8) Product Images from "A prototypical non-malignant epithelial model to study genome dynamics and concurrently monitor micro-RNAs and proteins in situ during oncogene-induced senescence"

Article Title: A prototypical non-malignant epithelial model to study genome dynamics and concurrently monitor micro-RNAs and proteins in situ during oncogene-induced senescence

Journal: BMC Genomics

doi: 10.1186/s12864-017-4375-1

Monitoring concurrently in situ miRs and proteins expression during OIS. Co-detection of miR34c and 5 3BP1 in senescent cells, employing the HBEC CDC6 Tet-ON system and our proposed protocol (see Additional file 17 and Additional file 18 : Figure S10). Multiple staining was performed in three consecutive states: in the OFF state where proliferation of HBECs is evident (“OFF”), 6 days after constitutive induction of CDC6 when cells are senescent (“6d ON”) and in the “escape from senescence” state termed “ESCAPED”. Step 1: miR34c FISH employing a double-DIG-labeled LNA probe, visualized as green emission in the cytoplasm, using TSA plus Fluorescein (emission at 518 nm). Step 2: GL13 staining, visualized at far red spectra as granules in the cytoplasm employing Alexa Fluor goat-anti-mouse (647 nm) (emission at 668 nm). Step 3: 53BP1 IF, visualized as red foci in the nucleus, employing Alexa Fluor goat-anti-mouse (568 nm) (emission at 618 nm). The specificity of each individual probe/antibody was tested by omitting sequentially the following reagents: miR34c probe, GL13 and anti-53BP1 antibodies at 6d ON cells. U6 snRNA and Scramble FISH, serving as positive and negative controls respectively, are presented in the Additional file 20 : Figure S12. Scale bar: 20 μm
Figure Legend Snippet: Monitoring concurrently in situ miRs and proteins expression during OIS. Co-detection of miR34c and 5 3BP1 in senescent cells, employing the HBEC CDC6 Tet-ON system and our proposed protocol (see Additional file 17 and Additional file 18 : Figure S10). Multiple staining was performed in three consecutive states: in the OFF state where proliferation of HBECs is evident (“OFF”), 6 days after constitutive induction of CDC6 when cells are senescent (“6d ON”) and in the “escape from senescence” state termed “ESCAPED”. Step 1: miR34c FISH employing a double-DIG-labeled LNA probe, visualized as green emission in the cytoplasm, using TSA plus Fluorescein (emission at 518 nm). Step 2: GL13 staining, visualized at far red spectra as granules in the cytoplasm employing Alexa Fluor goat-anti-mouse (647 nm) (emission at 668 nm). Step 3: 53BP1 IF, visualized as red foci in the nucleus, employing Alexa Fluor goat-anti-mouse (568 nm) (emission at 618 nm). The specificity of each individual probe/antibody was tested by omitting sequentially the following reagents: miR34c probe, GL13 and anti-53BP1 antibodies at 6d ON cells. U6 snRNA and Scramble FISH, serving as positive and negative controls respectively, are presented in the Additional file 20 : Figure S12. Scale bar: 20 μm

Techniques Used: In Situ, Expressing, Staining, Fluorescence In Situ Hybridization, Labeling

CDC6 induction resulted in R loop formation. a CDC6 induction increased total transcription levels, as measured by 5’-EU incorporation. Scale bar: 35 μm. b IF for S9.6, antibody specific for DNA:RNA hybrids, indicated increased possibility of R loop formation upon CDC6 expression. Concurrent IF detection of nucleolin, revealed nucleolus subcellular localization of the R loops. DNA:RNA hybrids disappeared after treatment with RNase A or RNase H showing the specificity of the reaction. Scale bar: 15 μm. c Double IF for 53BP1 and UBF showed re-localization of UBF from the nucleolar interior to the periphery associated with the perinucleolar distribution of 53BP1 foci (indicated by white arrows) reflecting DNA damage repair in heterochromatin-related structures [ 96 ]. Scale bar: 10 μm. d Heatmap of factors affecting R loops (Additional file 11 : Table S4) [ 98 ]
Figure Legend Snippet: CDC6 induction resulted in R loop formation. a CDC6 induction increased total transcription levels, as measured by 5’-EU incorporation. Scale bar: 35 μm. b IF for S9.6, antibody specific for DNA:RNA hybrids, indicated increased possibility of R loop formation upon CDC6 expression. Concurrent IF detection of nucleolin, revealed nucleolus subcellular localization of the R loops. DNA:RNA hybrids disappeared after treatment with RNase A or RNase H showing the specificity of the reaction. Scale bar: 15 μm. c Double IF for 53BP1 and UBF showed re-localization of UBF from the nucleolar interior to the periphery associated with the perinucleolar distribution of 53BP1 foci (indicated by white arrows) reflecting DNA damage repair in heterochromatin-related structures [ 96 ]. Scale bar: 10 μm. d Heatmap of factors affecting R loops (Additional file 11 : Table S4) [ 98 ]

Techniques Used: Expressing

In situ detection of miR34c in senescent cells. Detection of miR34c in senescent cells employing the HBEC CDC6 Tet-ON system. Double staining was performed in two consecutive states: in the OFF state where proliferation of HBECs is evident (“OFF”) and 6 days after constitutive induction of CDC6 when cells are senescent (“6d ON”). Step 1: Fluorescence FISH of miR34c employing a double-DIG-labeled LNA probe, visualized as green emission in the cytoplasm, using TSA plus Fluorescein (emitting at 518 nm). Step 2: GL13 staining, visualized at far red spectra as granules in the cytoplasm employing Alexa Fluor goat-anti-mouse (647 nm) (emitting at 668 nm). Scale bar: 50 μm
Figure Legend Snippet: In situ detection of miR34c in senescent cells. Detection of miR34c in senescent cells employing the HBEC CDC6 Tet-ON system. Double staining was performed in two consecutive states: in the OFF state where proliferation of HBECs is evident (“OFF”) and 6 days after constitutive induction of CDC6 when cells are senescent (“6d ON”). Step 1: Fluorescence FISH of miR34c employing a double-DIG-labeled LNA probe, visualized as green emission in the cytoplasm, using TSA plus Fluorescein (emitting at 518 nm). Step 2: GL13 staining, visualized at far red spectra as granules in the cytoplasm employing Alexa Fluor goat-anti-mouse (647 nm) (emitting at 668 nm). Scale bar: 50 μm

Techniques Used: In Situ, Double Staining, Fluorescence, Fluorescence In Situ Hybridization, Labeling, Staining

Transcriptome analysis of HBEC CDC6 Tet-ON cellular system. a Timeline of ECEE (epithelial cancer evolution experiment) showing time points where main biochemical and phenotypical events occur. High-throughput RNA sequencing analysis that was performed on 3-day induced (initiation of senescence phase) and in the “escaped” cells, compared to non-induced ones, revealed extensive alterations in the transciptome landscape. b Heatmap, showing hierarchical clustering, and ( c ) Venn diagram of the deregulated genes indicated that most of them were exclusive features of either of the two time points and not common ones nominating that they share different traits. d Adjustment of the transcriptome analysis to the “hallmarks” of cancer, utilizing Gene Ontology (GO) terms as shown in table, revealed that the “escaped” cells share the characteristic features of cancer cells. DDR/R refers to DNA damage response and repair pathways
Figure Legend Snippet: Transcriptome analysis of HBEC CDC6 Tet-ON cellular system. a Timeline of ECEE (epithelial cancer evolution experiment) showing time points where main biochemical and phenotypical events occur. High-throughput RNA sequencing analysis that was performed on 3-day induced (initiation of senescence phase) and in the “escaped” cells, compared to non-induced ones, revealed extensive alterations in the transciptome landscape. b Heatmap, showing hierarchical clustering, and ( c ) Venn diagram of the deregulated genes indicated that most of them were exclusive features of either of the two time points and not common ones nominating that they share different traits. d Adjustment of the transcriptome analysis to the “hallmarks” of cancer, utilizing Gene Ontology (GO) terms as shown in table, revealed that the “escaped” cells share the characteristic features of cancer cells. DDR/R refers to DNA damage response and repair pathways

Techniques Used: High Throughput Screening Assay, RNA Sequencing Assay

MiR analysis of HBEC CDC6 Tet-ON cellular system. a Principal component analysis of miR profiles showing that after escaping from the senescence programme, the cells reverted to the pre-induction stage, but considerable alterations of their miR expression persisted. b Hierarchical clustering of miRs that showed significant differences between the non-induced (OFF), induced (3d On and 6d On) and escaped from senescence (Escaped) cells
Figure Legend Snippet: MiR analysis of HBEC CDC6 Tet-ON cellular system. a Principal component analysis of miR profiles showing that after escaping from the senescence programme, the cells reverted to the pre-induction stage, but considerable alterations of their miR expression persisted. b Hierarchical clustering of miRs that showed significant differences between the non-induced (OFF), induced (3d On and 6d On) and escaped from senescence (Escaped) cells

Techniques Used: Expressing

Epithelial cancer evolution experiment (ECEE) in a CDC6-expressing non malignant epithelial model. a - b Generation and validation of the HBEC CDC6 Tet-ON cellular system. a Schematic representation of the lentiviral vectors (PLVX-Tet3G-BSD and PLVX-TRE3G-CDC6-BleoR) utilized for CDC6 transduction in HBECs (see details in “Methods” section). HBECs preserve their epithelial phenotype upon ectopic expression of hTERT and mutant CDK4 for immortalization. Inverted-phase contrast microscopy showed preservation of the epithelial morphology in HBECs upon transduction with lentiviruses, followed by treatment with antibiotics in order to isolate clones with inducible CDC6 over-expression. Scale bar: 10 μm. b Efficiency of CDC6 induction was confirmed both at protein (western blot) and mRNA (qRT-PCR) levels at the indicated time points. c Plot showing inverse relationship between proliferation rate, as measured by BrdU incorporation overnight, and senescent phenotype, as assessed by GL13 staining [ 53 ], during the time course of CDC6 induction. d Morphological and kinetic features observed by inverted-phase contrast microscopy (Scale bar: 20 μm), GL13 staining (Scale bar: 20 μm) and wound healing assay (Scale bar: 80 μm) at specific time points of ECEE representing normal, precancerous and cancerous stages of tumorigenesis. Non-induced cells (“OFF”) are near normal, 6-day induced cells recapitulate precancerous lesions, where senescent cells are evident (dark orange cells in cartoon [s]), and cells that have escaped from senescence (“ESCAPED”) share the invasive characteristics of cancer cells (dark green cells in cartoon [i]). A continuous counter-interaction between the oncogenic acting force and the anti-tumor reacting force (senescence) takes places at the precancerous stage, leading eventually to the prevalence of the tumor promoting effect, bypass of senescence and emergence of cancer cells [ 36 ]
Figure Legend Snippet: Epithelial cancer evolution experiment (ECEE) in a CDC6-expressing non malignant epithelial model. a - b Generation and validation of the HBEC CDC6 Tet-ON cellular system. a Schematic representation of the lentiviral vectors (PLVX-Tet3G-BSD and PLVX-TRE3G-CDC6-BleoR) utilized for CDC6 transduction in HBECs (see details in “Methods” section). HBECs preserve their epithelial phenotype upon ectopic expression of hTERT and mutant CDK4 for immortalization. Inverted-phase contrast microscopy showed preservation of the epithelial morphology in HBECs upon transduction with lentiviruses, followed by treatment with antibiotics in order to isolate clones with inducible CDC6 over-expression. Scale bar: 10 μm. b Efficiency of CDC6 induction was confirmed both at protein (western blot) and mRNA (qRT-PCR) levels at the indicated time points. c Plot showing inverse relationship between proliferation rate, as measured by BrdU incorporation overnight, and senescent phenotype, as assessed by GL13 staining [ 53 ], during the time course of CDC6 induction. d Morphological and kinetic features observed by inverted-phase contrast microscopy (Scale bar: 20 μm), GL13 staining (Scale bar: 20 μm) and wound healing assay (Scale bar: 80 μm) at specific time points of ECEE representing normal, precancerous and cancerous stages of tumorigenesis. Non-induced cells (“OFF”) are near normal, 6-day induced cells recapitulate precancerous lesions, where senescent cells are evident (dark orange cells in cartoon [s]), and cells that have escaped from senescence (“ESCAPED”) share the invasive characteristics of cancer cells (dark green cells in cartoon [i]). A continuous counter-interaction between the oncogenic acting force and the anti-tumor reacting force (senescence) takes places at the precancerous stage, leading eventually to the prevalence of the tumor promoting effect, bypass of senescence and emergence of cancer cells [ 36 ]

Techniques Used: Expressing, Transduction, Mutagenesis, Microscopy, Preserving, Clone Assay, Over Expression, Western Blot, Quantitative RT-PCR, BrdU Incorporation Assay, Staining, Wound Healing Assay

Phenotypic characterization of HBEC CDC6 Tet-ON system during ECEE. a Ultrastructural analysis of 6 days induced cells revealed flattened cellular morphology, with enlarged nucleus (N), enhanced protein-synthesis, as visualized by double nucleoli (n) and extensive Golgi apparatus (G), and formation of extracellular vesicles (v). Arrows depict extracellular vesicles. Scale bars 2 μm and 500 nm. b IF analysis of the proliferative marker (Cyclin A) and CDC6 showed double positive cells only after escape from senescence. Scale bar: 15 μm. c IF for an epithelial marker (E-cadherin) or a mesenchymal marker (Vimentin) along with CDC6 indicated EMT in the “escaped” cells. IF for CDC6 showed ubiquitous and uniform expression in the induced cells (“ON” and “ESCAPED”). Scale bar: 20 μm
Figure Legend Snippet: Phenotypic characterization of HBEC CDC6 Tet-ON system during ECEE. a Ultrastructural analysis of 6 days induced cells revealed flattened cellular morphology, with enlarged nucleus (N), enhanced protein-synthesis, as visualized by double nucleoli (n) and extensive Golgi apparatus (G), and formation of extracellular vesicles (v). Arrows depict extracellular vesicles. Scale bars 2 μm and 500 nm. b IF analysis of the proliferative marker (Cyclin A) and CDC6 showed double positive cells only after escape from senescence. Scale bar: 15 μm. c IF for an epithelial marker (E-cadherin) or a mesenchymal marker (Vimentin) along with CDC6 indicated EMT in the “escaped” cells. IF for CDC6 showed ubiquitous and uniform expression in the induced cells (“ON” and “ESCAPED”). Scale bar: 20 μm

Techniques Used: Marker, Expressing

DNA damage and DDR activation upon CDC6 induction are reduced in the “escaped” cells. a Cell cycle analysis upon CDC6 induction and plot of re-replicating cells (DNA content > 4n). b Comet assay showed DNA breaks in cells induced for the indicated time points. DNA damage was significantly reduced in the “escaped” cells. Plot depicts tail comet (moment) calculations. Red lines depict moment tails. Scale bar: 35 μm. c DDR activation in CDC6-induced cells as demonstrated by double IF for 53BP1 and CDC6 (Scale bar: 20 μm) along with western blotting for p53 and p21 WAF1/Cip1 . DDR is reduced in the “escaped” cells. Actin serves as a loading control.
Figure Legend Snippet: DNA damage and DDR activation upon CDC6 induction are reduced in the “escaped” cells. a Cell cycle analysis upon CDC6 induction and plot of re-replicating cells (DNA content > 4n). b Comet assay showed DNA breaks in cells induced for the indicated time points. DNA damage was significantly reduced in the “escaped” cells. Plot depicts tail comet (moment) calculations. Red lines depict moment tails. Scale bar: 35 μm. c DDR activation in CDC6-induced cells as demonstrated by double IF for 53BP1 and CDC6 (Scale bar: 20 μm) along with western blotting for p53 and p21 WAF1/Cip1 . DDR is reduced in the “escaped” cells. Actin serves as a loading control.

Techniques Used: Activation Assay, Cell Cycle Assay, Single Cell Gel Electrophoresis, Western Blot

9) Product Images from "Convergence of Protein Kinase C and JAK-STAT Signaling on Transcription Factor GATA-4"

Article Title: Convergence of Protein Kinase C and JAK-STAT Signaling on Transcription Factor GATA-4

Journal:

doi: 10.1128/MCB.25.22.9829-9844.2005

(A) GATA-4 contains a conserved PKC phosphorylation site within the C-terminal activation domain. The putative PKC phosphorylation site is shown in bold. (B) In vitro PKC phosphorylation of GST-GATA-4 fusion proteins containing either the N-(1-207) or
Figure Legend Snippet: (A) GATA-4 contains a conserved PKC phosphorylation site within the C-terminal activation domain. The putative PKC phosphorylation site is shown in bold. (B) In vitro PKC phosphorylation of GST-GATA-4 fusion proteins containing either the N-(1-207) or

Techniques Used: Activation Assay, In Vitro

In vivo association of the GATA-4 and STAT proteins with the ANF promoter. (A) Schematic representation of the various STAT and GATA elements on the ANF promoter. Putative elements correspond to in silico-identified consensus sequences. The location of
Figure Legend Snippet: In vivo association of the GATA-4 and STAT proteins with the ANF promoter. (A) Schematic representation of the various STAT and GATA elements on the ANF promoter. Putative elements correspond to in silico-identified consensus sequences. The location of

Techniques Used: In Vivo, In Silico

(A) Functional cooperation between GATA-4 and STAT1α is not restricted to the ANF promoter. NIH 3T3 cells were cotransfected with the −1176 VEGF, −360 c- fos , or −757 Bcl-x promoter-driven luciferase reporters and GATA-4,
Figure Legend Snippet: (A) Functional cooperation between GATA-4 and STAT1α is not restricted to the ANF promoter. NIH 3T3 cells were cotransfected with the −1176 VEGF, −360 c- fos , or −757 Bcl-x promoter-driven luciferase reporters and GATA-4,

Techniques Used: Functional Assay, Luciferase

(A) Mapping of the DNA elements required for GATA-4/STAT1α synergy. NIH 3T3 cells were cotransfected with the indicated ANF-luc constructs or a minimal BNP promoter driven by multimerized GATA binding sites (3xGATA) and with GATA-4, STAT1α,
Figure Legend Snippet: (A) Mapping of the DNA elements required for GATA-4/STAT1α synergy. NIH 3T3 cells were cotransfected with the indicated ANF-luc constructs or a minimal BNP promoter driven by multimerized GATA binding sites (3xGATA) and with GATA-4, STAT1α,

Techniques Used: Construct, Binding Assay

10) Product Images from "STAGA Recruits Mediator to the MYC Oncoprotein To Stimulate Transcription and Cell Proliferation ▿"

Article Title: STAGA Recruits Mediator to the MYC Oncoprotein To Stimulate Transcription and Cell Proliferation ▿

Journal:

doi: 10.1128/MCB.01402-07

Efficient interaction of MYC with core Mediator requires STAF65γ and an intact TAD. (A) STAF65γ-dependent association of STAGA and core Mediator subunits with endogenous MYC/MAX in HeLa cells. MAX was immunoprecipitated with a specific
Figure Legend Snippet: Efficient interaction of MYC with core Mediator requires STAF65γ and an intact TAD. (A) STAF65γ-dependent association of STAGA and core Mediator subunits with endogenous MYC/MAX in HeLa cells. MAX was immunoprecipitated with a specific

Techniques Used: Immunoprecipitation

11) Product Images from "Identification of pregnane-X receptor target genes and coactivator and corepressor binding to promoter elements in human hepatocytes"

Article Title: Identification of pregnane-X receptor target genes and coactivator and corepressor binding to promoter elements in human hepatocytes

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkn1047

Identification of PXR coactivators/corepressors binding to ( A ) CYP3A4, ( B ) CYP2B6, ( C ) MDR1, ( D ) UGT1A1 and ( E ) CYP4F12. Chromatin from cryopreserved human hepatocytes treated with vehicle (Control; 0.1% DMSO) or rifampicin (10 µM) for 3 h were immunoprecipitated with anti-GRIP1, -NCOR, -SRC3, -PGC1 α, -FKHR, -RIP140, -SMRT or -SHP antibody, and the enrichment of candidate coactivators/corepressors were determined by q-PCR. The error bars represent the SD from the mean of triplicate assays of an individual experiment, n = 3, * P
Figure Legend Snippet: Identification of PXR coactivators/corepressors binding to ( A ) CYP3A4, ( B ) CYP2B6, ( C ) MDR1, ( D ) UGT1A1 and ( E ) CYP4F12. Chromatin from cryopreserved human hepatocytes treated with vehicle (Control; 0.1% DMSO) or rifampicin (10 µM) for 3 h were immunoprecipitated with anti-GRIP1, -NCOR, -SRC3, -PGC1 α, -FKHR, -RIP140, -SMRT or -SHP antibody, and the enrichment of candidate coactivators/corepressors were determined by q-PCR. The error bars represent the SD from the mean of triplicate assays of an individual experiment, n = 3, * P

Techniques Used: Binding Assay, Immunoprecipitation, Polymerase Chain Reaction

12) Product Images from "Mechanism of Rapid Transcriptional Induction of Tumor Necrosis Factor Alpha-Responsive Genes by NF-?B"

Article Title: Mechanism of Rapid Transcriptional Induction of Tumor Necrosis Factor Alpha-Responsive Genes by NF-?B

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.22.18.6354-6362.2002

Model for transcriptional induction of NF-κB-responsive genes. In unstimulated cells, the promoters of NF-κB target genes are pre-occupied by the general transcription apparatus due to the function of a constitutively active transcription factor such as Sp1 that acts to recruit TFIID to the promoter. This complex is inadequate for multiple transcription rounds and thus can maintain only a low level of basal transcription. In stimulated cells, the association of NF-κB with the promoter is followed by multiple contacts with components of the transcription apparatus such as the TAF subunits, CBP, p300, and Mediator. These interactions promote multiple transcription cycles, resulting in high level of transcription.
Figure Legend Snippet: Model for transcriptional induction of NF-κB-responsive genes. In unstimulated cells, the promoters of NF-κB target genes are pre-occupied by the general transcription apparatus due to the function of a constitutively active transcription factor such as Sp1 that acts to recruit TFIID to the promoter. This complex is inadequate for multiple transcription rounds and thus can maintain only a low level of basal transcription. In stimulated cells, the association of NF-κB with the promoter is followed by multiple contacts with components of the transcription apparatus such as the TAF subunits, CBP, p300, and Mediator. These interactions promote multiple transcription cycles, resulting in high level of transcription.

Techniques Used:

Effect of NF-κB induction on promoter occupancy by the transcription apparatus. (A) CHIP assay using soluble chromatin extract from control or TNF-α-treated Jurkat cells and antibodies directed against TBP, TFIIB, Pol II, and the p65 component of NF-κB. The precipitated DNAs were used for PCR amplifications with primers spanning the core promoters of the indicated NF-κB target genes. (B) CHIP assays with antibodies directed against the coactivators CBP and p300 and analysis of A20 promoter by PCR. (C) CHIP assay with antibodies against the Mediator components Med7, Srb7, and TBP, which serve as a control. To confirm the amplification of the A20 promoter, the PCR products were analyzed by Southern blotting.
Figure Legend Snippet: Effect of NF-κB induction on promoter occupancy by the transcription apparatus. (A) CHIP assay using soluble chromatin extract from control or TNF-α-treated Jurkat cells and antibodies directed against TBP, TFIIB, Pol II, and the p65 component of NF-κB. The precipitated DNAs were used for PCR amplifications with primers spanning the core promoters of the indicated NF-κB target genes. (B) CHIP assays with antibodies directed against the coactivators CBP and p300 and analysis of A20 promoter by PCR. (C) CHIP assay with antibodies against the Mediator components Med7, Srb7, and TBP, which serve as a control. To confirm the amplification of the A20 promoter, the PCR products were analyzed by Southern blotting.

Techniques Used: Chromatin Immunoprecipitation, Polymerase Chain Reaction, Amplification, Southern Blot

13) Product Images from "IL-1?-specific recruitment of GCN5 histone acetyltransferase induces the release of PAF1 from chromatin for the de-repression of inflammatory response genes"

Article Title: IL-1?-specific recruitment of GCN5 histone acetyltransferase induces the release of PAF1 from chromatin for the de-repression of inflammatory response genes

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkt156

PAFc-associated PAF1 represses IL-1β–inducible genes. ( A ) Using data from Supplementary Figure S6B , the knockdown effect of individual PAFc components to IL-1β inducibility ( y -axis, relative ratio of PLAU mRNA with IL-1β to without IL-1β) and basal expression ( x -axis, relative ratio of PLAU mRNA with PAFc siRNA to control siRNA without IL-1β) are shown. ( B ) HepG2 cells were co-transfected with control or hPAF1 siRNA along with empty vector or indicated mPAFc expression vectors, and qRT–PCR for PLAU mRNA was performed. Bar indicates averages of data from three experiments; error bars represent SD. ( C ) ChIP analysis of CDC73 (as also known as parafibromin) and LEO1. HepG2 cells were stimulated with IL-1β (10 ng/ml) for 0 or 30 min. The specific protein occupancy at the target locus relative to the intergenic region was calculated and normalized to the IgG control. Bar indicates averages of data from two experiments; error bars represent standard deviation. ‘N.S.’ indicates not significant ( P > 0.05).
Figure Legend Snippet: PAFc-associated PAF1 represses IL-1β–inducible genes. ( A ) Using data from Supplementary Figure S6B , the knockdown effect of individual PAFc components to IL-1β inducibility ( y -axis, relative ratio of PLAU mRNA with IL-1β to without IL-1β) and basal expression ( x -axis, relative ratio of PLAU mRNA with PAFc siRNA to control siRNA without IL-1β) are shown. ( B ) HepG2 cells were co-transfected with control or hPAF1 siRNA along with empty vector or indicated mPAFc expression vectors, and qRT–PCR for PLAU mRNA was performed. Bar indicates averages of data from three experiments; error bars represent SD. ( C ) ChIP analysis of CDC73 (as also known as parafibromin) and LEO1. HepG2 cells were stimulated with IL-1β (10 ng/ml) for 0 or 30 min. The specific protein occupancy at the target locus relative to the intergenic region was calculated and normalized to the IgG control. Bar indicates averages of data from two experiments; error bars represent standard deviation. ‘N.S.’ indicates not significant ( P > 0.05).

Techniques Used: Expressing, Transfection, Plasmid Preparation, Quantitative RT-PCR, Chromatin Immunoprecipitation, Standard Deviation

14) Product Images from "Cyclic AMP-Stimulated Interaction between Steroidogenic Factor 1 and Diacylglycerol Kinase ? Facilitates Induction of CYP17 ▿"

Article Title: Cyclic AMP-Stimulated Interaction between Steroidogenic Factor 1 and Diacylglycerol Kinase ? Facilitates Induction of CYP17 ▿

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00355-07

cAMP increases DGK activity, and SF1 synergizes with DGK to stimulate CYP17 transcriptional activity. (A and B) H295R cells were treated for 5 min to 2 h with 1 mM Bt 2 cAMP and nuclear extracts purified for analysis of DGK activity using the OG-DG mixed-micelle assay (see Materials and Methods). Radiolabeled PA produced was resolved by TLC (representative plate shown in panel A) and quantified by phosphorimager scanning and densitometry (panel B). (C) Expression of DGK isoforms in H295R cells was determined by real-time RT-PCR of control RNA (50 ng) and the PCRs resolved on a 2% agarose gel. (D) Cells were transfected with pGL3-CYP17-2x57, pRL-TK, pCR3.1-SF1, and DGK expression plasmids and then treated for 16 h with 1 mM Bt 2 cAMP. Data graphed are normalized to Renilla activity (pRL-TK) and expressed as n -fold increases of pGL3-CYP17-2x57 activity over pRL-TK activity of the untreated control group. Data shown represent the means ± standard errors of the means from at least three separate experiments, each performed in triplicate.
Figure Legend Snippet: cAMP increases DGK activity, and SF1 synergizes with DGK to stimulate CYP17 transcriptional activity. (A and B) H295R cells were treated for 5 min to 2 h with 1 mM Bt 2 cAMP and nuclear extracts purified for analysis of DGK activity using the OG-DG mixed-micelle assay (see Materials and Methods). Radiolabeled PA produced was resolved by TLC (representative plate shown in panel A) and quantified by phosphorimager scanning and densitometry (panel B). (C) Expression of DGK isoforms in H295R cells was determined by real-time RT-PCR of control RNA (50 ng) and the PCRs resolved on a 2% agarose gel. (D) Cells were transfected with pGL3-CYP17-2x57, pRL-TK, pCR3.1-SF1, and DGK expression plasmids and then treated for 16 h with 1 mM Bt 2 cAMP. Data graphed are normalized to Renilla activity (pRL-TK) and expressed as n -fold increases of pGL3-CYP17-2x57 activity over pRL-TK activity of the untreated control group. Data shown represent the means ± standard errors of the means from at least three separate experiments, each performed in triplicate.

Techniques Used: Activity Assay, Purification, Produced, Thin Layer Chromatography, Expressing, Quantitative RT-PCR, Agarose Gel Electrophoresis, Transfection

15) Product Images from "Mutant p53 shapes the enhancer landscape of cancer cells in response to chronic immune signaling"

Article Title: Mutant p53 shapes the enhancer landscape of cancer cells in response to chronic immune signaling

Journal: Nature Communications

doi: 10.1038/s41467-017-01117-y

Chronic TNF-α signaling alters mutp53 and NFκB binding in colon cancer cells. a Heat maps of p53 R273H and NFκB/p65 ChIP-seq reads in SW480 cells treated with TNF-α for 0 or 16 h. Each row shows ±2 kb centered on p65 peaks, rank-ordered by the intensity of mutp53 and NFκB/p65 peaks and grouped by gained versus maintained mutp53 peaks. b De novo motif analyses of the TNF-gained and maintained mutp53 overlapping NFκB/p65 binding sites as noted in ( a ). c UCSC genome browser tracks of ChIP-seq signals for p53 R273H, NFκB/p65, H3K27ac, and H3K4me1 at the MMP9 , CCL2 , CYP24A1 , and CPA4 gene loci in untreated (purple) or TNF-α 16 h (pink)-treated SW480 cells. The y axis depicts the ChIP-seq signal and the x axis locates the genomic position with the enhancer regions highlighted in yellow. d Schematics of ChIP-qPCR amplicons and ChIP analyses with the indicated antibodies at the enhancers and non-specific regions of MMP9 , CCL2 , CYP24A1 , and CPA4 gene loci. ChIP-qPCR amplicons were designed to amplify the enhancer (A and B at MMP9 and A at CCL2 , CYP24A1 , and CPA4 ) or non-specific (C at MMP9 and B at CCL2 , CYP24A1 , and CPA4 ) regions of the target gene loci. ChIP experiments were performed using SW480 cells treated with TNF-α for 0, 8, 16, and 32 h. ChIPs for histone marks were normalized to H3. An average of two independent ChIP experiments that are representative of at least three is shown with error bars denoting the standard error. e Sequential ChIP (re-ChIP) with p53 antibody followed by IgG (control) and NFκB/p65 antibody performed in SW480 cells treated with TNF-α for 16 h. The ChIP-qPCR amplicons are identical to those used in ( d ). An average of two independent re-ChIP experiments that are representative of at least three is shown with error bars denoting the standard error
Figure Legend Snippet: Chronic TNF-α signaling alters mutp53 and NFκB binding in colon cancer cells. a Heat maps of p53 R273H and NFκB/p65 ChIP-seq reads in SW480 cells treated with TNF-α for 0 or 16 h. Each row shows ±2 kb centered on p65 peaks, rank-ordered by the intensity of mutp53 and NFκB/p65 peaks and grouped by gained versus maintained mutp53 peaks. b De novo motif analyses of the TNF-gained and maintained mutp53 overlapping NFκB/p65 binding sites as noted in ( a ). c UCSC genome browser tracks of ChIP-seq signals for p53 R273H, NFκB/p65, H3K27ac, and H3K4me1 at the MMP9 , CCL2 , CYP24A1 , and CPA4 gene loci in untreated (purple) or TNF-α 16 h (pink)-treated SW480 cells. The y axis depicts the ChIP-seq signal and the x axis locates the genomic position with the enhancer regions highlighted in yellow. d Schematics of ChIP-qPCR amplicons and ChIP analyses with the indicated antibodies at the enhancers and non-specific regions of MMP9 , CCL2 , CYP24A1 , and CPA4 gene loci. ChIP-qPCR amplicons were designed to amplify the enhancer (A and B at MMP9 and A at CCL2 , CYP24A1 , and CPA4 ) or non-specific (C at MMP9 and B at CCL2 , CYP24A1 , and CPA4 ) regions of the target gene loci. ChIP experiments were performed using SW480 cells treated with TNF-α for 0, 8, 16, and 32 h. ChIPs for histone marks were normalized to H3. An average of two independent ChIP experiments that are representative of at least three is shown with error bars denoting the standard error. e Sequential ChIP (re-ChIP) with p53 antibody followed by IgG (control) and NFκB/p65 antibody performed in SW480 cells treated with TNF-α for 16 h. The ChIP-qPCR amplicons are identical to those used in ( d ). An average of two independent re-ChIP experiments that are representative of at least three is shown with error bars denoting the standard error

Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

Mutp53 enhancer binding is positively correlated with enhancer transcription. a UCSC genome browser images for MMP9 and CPA4 gene loci showing the ChIP-seq signal for mutp53 binding and GRO-seq peaks with the enhancer regions highlighted in yellow. b Boxplots showing log 2 fold change in response to TNF-α in GRO-seq signal of nascent transcripts centered upon intergenic gained or maintained mutp53 peaks that overlap with NFκB and H3K27ac peaks. c Analyses of (left) GRO-seq reads per bp per intergenic ChIP-seq peaks of mutp53 and NFκB/p65, as indicated, and (right) GRO-seq reads per bp per TSS at promoters closest to intergenic ChIP-seq peaks defined in the left panel. d qRT-PCR analyses of the indicated eRNAs and mRNAs in SW480 cells and e HCT116 cells treated with TNF-α for 0, 8, or 16 h. The expression levels shown after TNF-α treatment are relative to the levels before treatment. The bar graphs represent the average of three independent experiments with the error bars denoting the standard error
Figure Legend Snippet: Mutp53 enhancer binding is positively correlated with enhancer transcription. a UCSC genome browser images for MMP9 and CPA4 gene loci showing the ChIP-seq signal for mutp53 binding and GRO-seq peaks with the enhancer regions highlighted in yellow. b Boxplots showing log 2 fold change in response to TNF-α in GRO-seq signal of nascent transcripts centered upon intergenic gained or maintained mutp53 peaks that overlap with NFκB and H3K27ac peaks. c Analyses of (left) GRO-seq reads per bp per intergenic ChIP-seq peaks of mutp53 and NFκB/p65, as indicated, and (right) GRO-seq reads per bp per TSS at promoters closest to intergenic ChIP-seq peaks defined in the left panel. d qRT-PCR analyses of the indicated eRNAs and mRNAs in SW480 cells and e HCT116 cells treated with TNF-α for 0, 8, or 16 h. The expression levels shown after TNF-α treatment are relative to the levels before treatment. The bar graphs represent the average of three independent experiments with the error bars denoting the standard error

Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Quantitative RT-PCR, Expressing

Mutp53 and NFκB interact and impact each other’s binding at enhancers. a Purified wild-type and mutp53 proteins bind directly to purified NFκB/p65 as revealed by immunoblot analysis with an antibody that recognizes p65. Input samples for the p53 proteins were also analyzed by immunoblot analysis with an antibody that recognizes both wild-type and mutp53. Three independent interaction assays were performed. b ChIP-qPCR analyses of NFκB/p65 and p53 R273H at the enhancer (A and B at MMP9 and A at CCL2 , CYP24A1 , and CPA4 ) or non-specific (C at MMP9 and B at CCL2 , CYP24A1 , and CPA4 ) regions of the target gene loci in SW480 cells transfected with non-targeting control or p65 siRNA and following TNF-α treatment for 0 or 16 h. c ChIP-qPCR analyses of p53 R273H and NFκB/p65 binding at identical genomic regions examined in ( b ). The SW480 cells were induced to express LacZ (control) or p53 (p53) shRNA and treated with TNF-α for 0 or 16 h. For both ChIP experiments, an average of two independent experiments that are representative of at least three is shown with error bars denoting the standard error
Figure Legend Snippet: Mutp53 and NFκB interact and impact each other’s binding at enhancers. a Purified wild-type and mutp53 proteins bind directly to purified NFκB/p65 as revealed by immunoblot analysis with an antibody that recognizes p65. Input samples for the p53 proteins were also analyzed by immunoblot analysis with an antibody that recognizes both wild-type and mutp53. Three independent interaction assays were performed. b ChIP-qPCR analyses of NFκB/p65 and p53 R273H at the enhancer (A and B at MMP9 and A at CCL2 , CYP24A1 , and CPA4 ) or non-specific (C at MMP9 and B at CCL2 , CYP24A1 , and CPA4 ) regions of the target gene loci in SW480 cells transfected with non-targeting control or p65 siRNA and following TNF-α treatment for 0 or 16 h. c ChIP-qPCR analyses of p53 R273H and NFκB/p65 binding at identical genomic regions examined in ( b ). The SW480 cells were induced to express LacZ (control) or p53 (p53) shRNA and treated with TNF-α for 0 or 16 h. For both ChIP experiments, an average of two independent experiments that are representative of at least three is shown with error bars denoting the standard error

Techniques Used: Binding Assay, Purification, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Transfection, shRNA

16) Product Images from "Heme regulates the dynamic exchange of Bach1 and NF-E2-related factors in the Maf transcription factor network"

Article Title: Heme regulates the dynamic exchange of Bach1 and NF-E2-related factors in the Maf transcription factor network

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.0308083100

Competitive binding of Bach1 and Nrf2 regulates RNA polymerase recruitment and promoter clearance. ( A ) NIH 3T3 cells were treated with 10 μM hemin for 2 h, washed, and cultured further in hemin-free medium. Expression of HO-1 mRNA was determined
Figure Legend Snippet: Competitive binding of Bach1 and Nrf2 regulates RNA polymerase recruitment and promoter clearance. ( A ) NIH 3T3 cells were treated with 10 μM hemin for 2 h, washed, and cultured further in hemin-free medium. Expression of HO-1 mRNA was determined

Techniques Used: Binding Assay, Cell Culture, Expressing

17) Product Images from "BIX01294, an inhibitor of histone methyltransferase, induces autophagy-dependent differentiation of glioma stem-like cells"

Article Title: BIX01294, an inhibitor of histone methyltransferase, induces autophagy-dependent differentiation of glioma stem-like cells

Journal: Scientific Reports

doi: 10.1038/srep38723

BIX01294-induced autophagy requires ATG proteins. Knockdown of ATG5, ATG7, ULK1 and BECN1 at the mRNA ( A ) and protein level ( B ) using specific siRNAs. Glioma cells were transfected with ATG -targeting siRNA for 48 h, followed by 2 μM BIX01294 for 24 h. ( A ) Each bar represents the mean ± SEM of three independent experiments. Statistical significance calculated to siCtrl-transfected cells (*P
Figure Legend Snippet: BIX01294-induced autophagy requires ATG proteins. Knockdown of ATG5, ATG7, ULK1 and BECN1 at the mRNA ( A ) and protein level ( B ) using specific siRNAs. Glioma cells were transfected with ATG -targeting siRNA for 48 h, followed by 2 μM BIX01294 for 24 h. ( A ) Each bar represents the mean ± SEM of three independent experiments. Statistical significance calculated to siCtrl-transfected cells (*P

Techniques Used: Transfection

18) Product Images from "Target genes of the largest human SWI/SNF complex subunit control cell growth"

Article Title: Target genes of the largest human SWI/SNF complex subunit control cell growth

Journal: The Biochemical journal

doi: 10.1042/BJ20101358

c- myc mRNA levels are reduced after the expression of hOsa2, but increased after the knockdown of endogenous hOsa2
Figure Legend Snippet: c- myc mRNA levels are reduced after the expression of hOsa2, but increased after the knockdown of endogenous hOsa2

Techniques Used: Expressing

hOsa2 represses c- myc and activates p21 gene transcription
Figure Legend Snippet: hOsa2 represses c- myc and activates p21 gene transcription

Techniques Used:

Induced expression of hOsa2 results in impaired cell growth and accumulation of cells in the G 1 phase
Figure Legend Snippet: Induced expression of hOsa2 results in impaired cell growth and accumulation of cells in the G 1 phase

Techniques Used: Expressing

Induced expression of hOsa2 decreases BrdU incorporation
Figure Legend Snippet: Induced expression of hOsa2 decreases BrdU incorporation

Techniques Used: Expressing, BrdU Incorporation Assay

Changes in p53, p21 and c-Myc protein levels after induction of hOsa2
Figure Legend Snippet: Changes in p53, p21 and c-Myc protein levels after induction of hOsa2

Techniques Used:

Activation of the p21 promoter upon co-expression of hOsa2
Figure Legend Snippet: Activation of the p21 promoter upon co-expression of hOsa2

Techniques Used: Activation Assay, Expressing

Purification of protein complexes associated with hOsa2 and hOsa2ΔARID
Figure Legend Snippet: Purification of protein complexes associated with hOsa2 and hOsa2ΔARID

Techniques Used: Purification

19) Product Images from "The G2/M Regulator Histone Demethylase PHF8 Is Targeted for Degradation by the Anaphase-Promoting Complex Containing CDC20"

Article Title: The G2/M Regulator Histone Demethylase PHF8 Is Targeted for Degradation by the Anaphase-Promoting Complex Containing CDC20

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00689-13

PHF8 interacts with the APC cdc20 during M phase. (A and B) MCF7-pOZ-Flag-HA-PHF8 cells were used for complex purification. Tandem affinity purification (Flag followed by HA IPs) was carried out on nuclear extracts. Immunocomplexes were subjected to silver
Figure Legend Snippet: PHF8 interacts with the APC cdc20 during M phase. (A and B) MCF7-pOZ-Flag-HA-PHF8 cells were used for complex purification. Tandem affinity purification (Flag followed by HA IPs) was carried out on nuclear extracts. Immunocomplexes were subjected to silver

Techniques Used: Purification, Affinity Purification

The PHF8 LPKLF mutant resists polyubiquitylation by the APC cdc20 and proteasomal degradation but has an as-yet-undefined effect on cells. (A) WT or mutant Flag-HA-PHF8 with either the entire 7-amino-acid motif deleted (Δ495–501) or each
Figure Legend Snippet: The PHF8 LPKLF mutant resists polyubiquitylation by the APC cdc20 and proteasomal degradation but has an as-yet-undefined effect on cells. (A) WT or mutant Flag-HA-PHF8 with either the entire 7-amino-acid motif deleted (Δ495–501) or each

Techniques Used: Mutagenesis

A novel motif on PHF8 governs its binding to CDC20. (A) The D-box motif on PHF8 was mutated in Flag-HA-PHF8 to generate mutants with an R481A, L484A, or N488A mutation or the RLN-Triple mutant (R481A L484A N488A triple mutant). The empty vector, wild-type
Figure Legend Snippet: A novel motif on PHF8 governs its binding to CDC20. (A) The D-box motif on PHF8 was mutated in Flag-HA-PHF8 to generate mutants with an R481A, L484A, or N488A mutation or the RLN-Triple mutant (R481A L484A N488A triple mutant). The empty vector, wild-type

Techniques Used: Binding Assay, Mutagenesis, Plasmid Preparation

PHF8 protein levels can be modulated by changing CDC20 levels. (A) The empty vector control or increasing amounts of HA-CDC20 (0.2, 0.5, and 1.0 μg) were transfected into HEK293T cells, and lysates were immunoblotted for endogenous PHF8. (B) A
Figure Legend Snippet: PHF8 protein levels can be modulated by changing CDC20 levels. (A) The empty vector control or increasing amounts of HA-CDC20 (0.2, 0.5, and 1.0 μg) were transfected into HEK293T cells, and lysates were immunoblotted for endogenous PHF8. (B) A

Techniques Used: Plasmid Preparation, Transfection

20) Product Images from "New stably transfected bioluminescent cells expressing FLAG epitope-tagged estrogen receptors to study their chromatin recruitment"

Article Title: New stably transfected bioluminescent cells expressing FLAG epitope-tagged estrogen receptors to study their chromatin recruitment

Journal: BMC Biotechnology

doi: 10.1186/1472-6750-9-77

Compared efficiencies of FLAG-ERα/β immunoprecipitations . A) Soluble chromatin was prepared from HELN-Fα or HELN-Fβ cells stimulated 1 hr with 10 nM of E2, either by using chromatin cross-linked with the same procedure as that used for kinetic ChIP experiments (named
Figure Legend Snippet: Compared efficiencies of FLAG-ERα/β immunoprecipitations . A) Soluble chromatin was prepared from HELN-Fα or HELN-Fβ cells stimulated 1 hr with 10 nM of E2, either by using chromatin cross-linked with the same procedure as that used for kinetic ChIP experiments (named "+cross"), or by using uncrosslinked chromatin solubilized in a mild buffer (named "-cross"). Then, 40 μg of ± cross-linked chromatin were subjected to a 10% slab gel electrophoresis before (Inputs) or after (IP) immunoprecipitation with the anti-FLAG antibody. B) HELN-Fα or HELN-Fβ cells were stimulated 1 hr with 10 nM of E2, then, soluble chromatin preparation and immunoprecipitation (with the HC20 anti ERα antibody) were performed by using an identical procedure than that used for ChIP-kinetics experiments. Real time PCR quantification of either IP chromatin or input was performed at each incubation time. Amplified signals from IP chromatin were calculated as the percentage of amplified input signals obtained during the same amplification. Corresponding values plotted at indicated time were expressed as a percentage of the maximum value obtained in this experiment. Values are mean ± SD of two independent immunoprecipitation assays.

Techniques Used: Chromatin Immunoprecipitation, Nucleic Acid Electrophoresis, Immunoprecipitation, Real-time Polymerase Chain Reaction, Incubation, Amplification

Recruitment of ERα/β to the promoter of the luciferase transgene in HELN-Fα/β cells . Kinetic ChIP experiments were performed using the anti-FLAG antibody. HELN-Fα ( A ) or HELN-Fβ ( B ) cells were cultured in 3% dextran-charcoal treated FCS. Twenty four hours before experiment, they were deprived of serum and subsequently treated for 2 hr with 2.5 μM α-amanitin, and then with 10 nM E2. Cells were cross-linked at indicated times. Soluble chromatin was prepared on sampled cells at indicated times as described in material and methods. Real time PCR quantification of either IP chromatin or input was performed at each incubation time. Amplified signals from IP chromatin were calculated as the percentage of amplified input signals obtained during the same amplification. Corresponding values plotted for one curve were expressed as the percentage of the maximum value of % input (% input max) obtained for that curve and for one IP. One ChIP kinetic curve shown is representative of at least two independent experiments and values are mean ± SD of two independent immunoprecipitation assays using the same preparation of chromatin. The % input max average value (corresponding to the two IPs) of one curve may fluctuate among different independent experiments corresponding to the same kinetic. The corresponding amplitudes of variation are: in A (HELN-Fα) Imin = 2.1, Imax = 4.3; in B (HELNβ) Imin = 1.4 Imax = 3.5. Flag-ERα (A) or Flag-ERβ (B) protein levels were analyzed by Western blot experiments. Cell treatments for ChiP assays and Western blot experiments were identical. Extracts were prepared at indicated times, and Western blotted with antibodies for FLAG (F3165) or actin.
Figure Legend Snippet: Recruitment of ERα/β to the promoter of the luciferase transgene in HELN-Fα/β cells . Kinetic ChIP experiments were performed using the anti-FLAG antibody. HELN-Fα ( A ) or HELN-Fβ ( B ) cells were cultured in 3% dextran-charcoal treated FCS. Twenty four hours before experiment, they were deprived of serum and subsequently treated for 2 hr with 2.5 μM α-amanitin, and then with 10 nM E2. Cells were cross-linked at indicated times. Soluble chromatin was prepared on sampled cells at indicated times as described in material and methods. Real time PCR quantification of either IP chromatin or input was performed at each incubation time. Amplified signals from IP chromatin were calculated as the percentage of amplified input signals obtained during the same amplification. Corresponding values plotted for one curve were expressed as the percentage of the maximum value of % input (% input max) obtained for that curve and for one IP. One ChIP kinetic curve shown is representative of at least two independent experiments and values are mean ± SD of two independent immunoprecipitation assays using the same preparation of chromatin. The % input max average value (corresponding to the two IPs) of one curve may fluctuate among different independent experiments corresponding to the same kinetic. The corresponding amplitudes of variation are: in A (HELN-Fα) Imin = 2.1, Imax = 4.3; in B (HELNβ) Imin = 1.4 Imax = 3.5. Flag-ERα (A) or Flag-ERβ (B) protein levels were analyzed by Western blot experiments. Cell treatments for ChiP assays and Western blot experiments were identical. Extracts were prepared at indicated times, and Western blotted with antibodies for FLAG (F3165) or actin.

Techniques Used: Luciferase, Chromatin Immunoprecipitation, Cell Culture, Real-time Polymerase Chain Reaction, Incubation, Amplification, Immunoprecipitation, Western Blot

Recruitment of ERα and Pol II to the pS2 promoter in MCF-7 cells . ChIP-kinetic experiments were performed using anti ERα antibody (HC-20) ( A ) or anti-Pol II antibody ( B ). Cells were cultured in 10% FCS and then in 3% dextran-charcoal treated FCS for three days. Twenty four hours before experiment, cells were deprived of serum and subsequently treated for 2 hr with 2.5 μM α-amanitin, and then with 10 nM E2 during 8 hours. Chromatin was prepared as in Figure 4 or 5. Values are mean ± SD of two independent immunoprecipitation assays of the same chromatin, and expressed as in Figure 4. ERα (A) protein levels were analyzed by Western blot experiments. MCF-7 treatments for ChiP assays and Western blot experiments were identical. Extracts were prepared at indicated times, and Western blotted with antibodies for ERα (HC-20) or actin.
Figure Legend Snippet: Recruitment of ERα and Pol II to the pS2 promoter in MCF-7 cells . ChIP-kinetic experiments were performed using anti ERα antibody (HC-20) ( A ) or anti-Pol II antibody ( B ). Cells were cultured in 10% FCS and then in 3% dextran-charcoal treated FCS for three days. Twenty four hours before experiment, cells were deprived of serum and subsequently treated for 2 hr with 2.5 μM α-amanitin, and then with 10 nM E2 during 8 hours. Chromatin was prepared as in Figure 4 or 5. Values are mean ± SD of two independent immunoprecipitation assays of the same chromatin, and expressed as in Figure 4. ERα (A) protein levels were analyzed by Western blot experiments. MCF-7 treatments for ChiP assays and Western blot experiments were identical. Extracts were prepared at indicated times, and Western blotted with antibodies for ERα (HC-20) or actin.

Techniques Used: Chromatin Immunoprecipitation, Cell Culture, Immunoprecipitation, Western Blot

Structure, expression and binding properties of the FLAG-ERα/β proteins . A) Representation of FLAG-ERα/β proteins showing the amino acid sequence length of ERα and ERβ, as well as their different domains, and the position of the FLAG tag. B) Western blot using the M2 anti-FLAG antibody to probe the relative amounts of FLAG-ERα or FLAG-ERβ present in 40 μg of total protein extracts prepared from HELN-Fα or HELN-Fβ stable cell lines cultured in 3% DCC (in absence of phenol red). C) Scatchard plot analysis of specific binding of [3H]-E2 to FLAG-ERα or FLAG-ERβ during 6 hours in a whole cell assay. Dissociation constant (Kd) is indicated.
Figure Legend Snippet: Structure, expression and binding properties of the FLAG-ERα/β proteins . A) Representation of FLAG-ERα/β proteins showing the amino acid sequence length of ERα and ERβ, as well as their different domains, and the position of the FLAG tag. B) Western blot using the M2 anti-FLAG antibody to probe the relative amounts of FLAG-ERα or FLAG-ERβ present in 40 μg of total protein extracts prepared from HELN-Fα or HELN-Fβ stable cell lines cultured in 3% DCC (in absence of phenol red). C) Scatchard plot analysis of specific binding of [3H]-E2 to FLAG-ERα or FLAG-ERβ during 6 hours in a whole cell assay. Dissociation constant (Kd) is indicated.

Techniques Used: Expressing, Binding Assay, Sequencing, FLAG-tag, Western Blot, Stable Transfection, Cell Culture, Droplet Countercurrent Chromatography

21) Product Images from "The C-Terminal Transactivation Domain of STAT1 Has a Gene-Specific Role in Transactivation and Cofactor Recruitment"

Article Title: The C-Terminal Transactivation Domain of STAT1 Has a Gene-Specific Role in Transactivation and Cofactor Recruitment

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.02879

Recruitment and phosphorylation of Pol II at Irf1 gene and occupancy of TFIIH, p-TEFb, and Mediator components at the Irf1 TSS. (A) Schematic representation of Pol II, GTFs, and the Mediator complex at a GAS-driven gene promoter. Components of the Mediator, TFIIH, and p-TEFb complexes analyzed by ChIP are indicated. (B–L) BMDMs from WT and Stat1 β/β mice were stimulated with IFNγ for the times indicated or left untreated (0 h). Association of Pol II, S5pPol II, and S2pPol II with the Irf1 promoter around the TSS (B–D) and of S2pPol II within the Irf1 gene body (E) . Association of ERCC3 (F) and CDK9 (G) at the Irf1 TSS and of MED18 (H) , MED4 (I) , MED24 (J) , MED26 (K) , and MED1 (L) at the Irf1 GAS. Data were normalized to the input control. Mean values ± SE from two (H) or three (all others) independent experiments are shown; * P ≤ 0.05 , ** P ≤ 0.01 , *** P ≤ 0.001 .
Figure Legend Snippet: Recruitment and phosphorylation of Pol II at Irf1 gene and occupancy of TFIIH, p-TEFb, and Mediator components at the Irf1 TSS. (A) Schematic representation of Pol II, GTFs, and the Mediator complex at a GAS-driven gene promoter. Components of the Mediator, TFIIH, and p-TEFb complexes analyzed by ChIP are indicated. (B–L) BMDMs from WT and Stat1 β/β mice were stimulated with IFNγ for the times indicated or left untreated (0 h). Association of Pol II, S5pPol II, and S2pPol II with the Irf1 promoter around the TSS (B–D) and of S2pPol II within the Irf1 gene body (E) . Association of ERCC3 (F) and CDK9 (G) at the Irf1 TSS and of MED18 (H) , MED4 (I) , MED24 (J) , MED26 (K) , and MED1 (L) at the Irf1 GAS. Data were normalized to the input control. Mean values ± SE from two (H) or three (all others) independent experiments are shown; * P ≤ 0.05 , ** P ≤ 0.01 , *** P ≤ 0.001 .

Techniques Used: Chromatin Immunoprecipitation, Mouse Assay

22) Product Images from "Evidence for Multiple Mediator Complexes in Yeast Independently Recruited by Activated Heat Shock Factor"

Article Title: Evidence for Multiple Mediator Complexes in Yeast Independently Recruited by Activated Heat Shock Factor

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00005-16

Anchoring Med15 obviates core Mediator recruitment but not that of the CKM, while anchoring Hsf1 obviates both. (A) ChIP analysis of Med15 AA strain YM125 conducted and quantified as in . Untagged subunits were detected using antibodies raised against
Figure Legend Snippet: Anchoring Med15 obviates core Mediator recruitment but not that of the CKM, while anchoring Hsf1 obviates both. (A) ChIP analysis of Med15 AA strain YM125 conducted and quantified as in . Untagged subunits were detected using antibodies raised against

Techniques Used: Chromatin Immunoprecipitation

23) Product Images from "Brd4 bridges the transcriptional regulators, Aire and P-TEFb, to promote elongation of peripheral-tissue antigen transcripts in thymic stromal cells"

Article Title: Brd4 bridges the transcriptional regulators, Aire and P-TEFb, to promote elongation of peripheral-tissue antigen transcripts in thymic stromal cells

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1512081112

A model for Brd4-dependent interaction between Aire and P-TEFb. After Aire is phosphorylated at T69 by DNA-PK, CBP is recruited and acetylates Aire on CARD lysine residues. These residues are recognized by Brd4’s amino-terminal bromodomain, BD1, corecruiting P-TEFb, which releases promoter-proximal Pol-II pausing, promoting transcriptional elongation and splicing.
Figure Legend Snippet: A model for Brd4-dependent interaction between Aire and P-TEFb. After Aire is phosphorylated at T69 by DNA-PK, CBP is recruited and acetylates Aire on CARD lysine residues. These residues are recognized by Brd4’s amino-terminal bromodomain, BD1, corecruiting P-TEFb, which releases promoter-proximal Pol-II pausing, promoting transcriptional elongation and splicing.

Techniques Used:

24) Product Images from "Distinct steady-state nuclear receptor coregulator complexes exist invivo"

Article Title: Distinct steady-state nuclear receptor coregulator complexes exist invivo

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi:

High molecular mass complexes contain CBP and RNA pol II. Fractionation of T47D lysate on a Superose 6 column was analyzed by immunoblot with CBP and RNA Pol II-specific antibodies (CBP and RNA pol II). Recombinant baculovirus-expressed CBP also was fractionated (CBP BAC). Indicated are elution peaks of molecular mass markers: mammalian SWI/SNF complex (≈2 MDa) and thyroglobulin (670 kDa). The void volume (4 MDa for globular proteins) was determined at fraction 20 by silver staining after fractionation of T47D cell lysate (data not shown).
Figure Legend Snippet: High molecular mass complexes contain CBP and RNA pol II. Fractionation of T47D lysate on a Superose 6 column was analyzed by immunoblot with CBP and RNA Pol II-specific antibodies (CBP and RNA pol II). Recombinant baculovirus-expressed CBP also was fractionated (CBP BAC). Indicated are elution peaks of molecular mass markers: mammalian SWI/SNF complex (≈2 MDa) and thyroglobulin (670 kDa). The void volume (4 MDa for globular proteins) was determined at fraction 20 by silver staining after fractionation of T47D cell lysate (data not shown).

Techniques Used: Fractionation, Recombinant, BAC Assay, Multiple Displacement Amplification, Silver Staining

25) Product Images from "miR675 Accelerates Malignant Transformation of Mesenchymal Stem Cells by Blocking DNA Mismatch Repair"

Article Title: miR675 Accelerates Malignant Transformation of Mesenchymal Stem Cells by Blocking DNA Mismatch Repair

Journal: Molecular Therapy. Nucleic Acids

doi: 10.1016/j.omtn.2018.11.010

miR675 Triggers MSI and Abnormal Gene Expression in the Human Mesenchymal Stem Cells Infected with rLV and rLV-miR675, Respectively (A) Microsatellite instability (MSI) analysis through dot blot (slot blot) using various biotin-labeling MSI probes (biotin-MSIs). (B) (a) Chromosome conformation capture (3C)-chromatin immunoprecipitation (ChIP) with anti-P300 and anti-Pol II. The chromatin was cross-linked, digested with restriction enzymes, and ligated under conditions that favor intramolecular ligation. Immediately after ligation, the chromatin was immunoprecipitated using an antibody (anti-P300, anti-Pol II) against the protein of interest. Thereafter, the cross-links were reversed and the DNA was purified further. The PCR anlysis was applied for detecting CyclinD1 promoter-enhancer coupling product using CyclinD1 promoter and enhancer primers. The CyclinD1 promoter and enhancer was the INPUT. (b) The quantitative analysis of ChIP-3C. (C) (a) Western blotting with anti-Rad51, anti-CDK2, anti-CyclinE, anti-CDK4, anti-CyclinD1, anti-PCNA, anti-ppRB, anti-E2F1, anti-P18, anti-P21, anti-PKM2, anti-c-Myc, and anti-Chk1. β-actin was the internal control. (b) The gray scan analysis of positive bands of western blotting.
Figure Legend Snippet: miR675 Triggers MSI and Abnormal Gene Expression in the Human Mesenchymal Stem Cells Infected with rLV and rLV-miR675, Respectively (A) Microsatellite instability (MSI) analysis through dot blot (slot blot) using various biotin-labeling MSI probes (biotin-MSIs). (B) (a) Chromosome conformation capture (3C)-chromatin immunoprecipitation (ChIP) with anti-P300 and anti-Pol II. The chromatin was cross-linked, digested with restriction enzymes, and ligated under conditions that favor intramolecular ligation. Immediately after ligation, the chromatin was immunoprecipitated using an antibody (anti-P300, anti-Pol II) against the protein of interest. Thereafter, the cross-links were reversed and the DNA was purified further. The PCR anlysis was applied for detecting CyclinD1 promoter-enhancer coupling product using CyclinD1 promoter and enhancer primers. The CyclinD1 promoter and enhancer was the INPUT. (b) The quantitative analysis of ChIP-3C. (C) (a) Western blotting with anti-Rad51, anti-CDK2, anti-CyclinE, anti-CDK4, anti-CyclinD1, anti-PCNA, anti-ppRB, anti-E2F1, anti-P18, anti-P21, anti-PKM2, anti-c-Myc, and anti-Chk1. β-actin was the internal control. (b) The gray scan analysis of positive bands of western blotting.

Techniques Used: Expressing, Infection, Dot Blot, Labeling, Chromatin Immunoprecipitation, Ligation, Immunoprecipitation, Purification, Polymerase Chain Reaction, Western Blot

26) Product Images from "Heterochromatin assembly and transcriptome repression by Set1 in coordination with a class II histone deacetylase"

Article Title: Heterochromatin assembly and transcriptome repression by Set1 in coordination with a class II histone deacetylase

Journal: eLife

doi: 10.7554/eLife.04506

Model for Set1 functions at euchromatin and heterochromatin domains. At euchromatin domains, the Set1C/COMPASS complex is recruited to active Pol II genes and provides the H3K4me marks. Set1 is also recruited to certain lowly expressed and repressed genes associated with developmental and stress-response pathways in part by Atf1, other transcription factors (TFs), and probably transcriptionally poised Pol II. Set1 acts in a parallel pathway with the histone deacetylase (HDAC) Clr3 to impose transcriptional repression at these loci. At a heterochromatin domain such as the pericentromeric region, Atf1 and probably other unidentified TFs mediate the recruitment of Set1 to sites enriched for tRNAs known to act as boundary elements. Set1 coordinates with Clr3 in the establishment of SUV39H1/Clr4-mediated H3K9me/HP1 (HP: Swi6 and Chp2) heterochromatin and suppression of bidirectional transcription independently of H3K4me and the other Set1C subunits. Set1-mediated silencing could occur via methylation of nonhistone substrate(s) through the same or different pathways from those of RNAi (i.e., RITS, Rdp1, Dicer) or the exosome (not shown). DOI: http://dx.doi.org/10.7554/eLife.04506.024
Figure Legend Snippet: Model for Set1 functions at euchromatin and heterochromatin domains. At euchromatin domains, the Set1C/COMPASS complex is recruited to active Pol II genes and provides the H3K4me marks. Set1 is also recruited to certain lowly expressed and repressed genes associated with developmental and stress-response pathways in part by Atf1, other transcription factors (TFs), and probably transcriptionally poised Pol II. Set1 acts in a parallel pathway with the histone deacetylase (HDAC) Clr3 to impose transcriptional repression at these loci. At a heterochromatin domain such as the pericentromeric region, Atf1 and probably other unidentified TFs mediate the recruitment of Set1 to sites enriched for tRNAs known to act as boundary elements. Set1 coordinates with Clr3 in the establishment of SUV39H1/Clr4-mediated H3K9me/HP1 (HP: Swi6 and Chp2) heterochromatin and suppression of bidirectional transcription independently of H3K4me and the other Set1C subunits. Set1-mediated silencing could occur via methylation of nonhistone substrate(s) through the same or different pathways from those of RNAi (i.e., RITS, Rdp1, Dicer) or the exosome (not shown). DOI: http://dx.doi.org/10.7554/eLife.04506.024

Techniques Used: Histone Deacetylase Assay, Activated Clotting Time Assay, Methylation

Derepression of ste11 in mutants deficient in both atf1 and set1 . Expression changes on forward and reverse strands at the ste11 locus in atf1Δ (blue dashed lines) , set1Δ (red solid lines), and atf1Δ set1Δ (dotted purple lines) mutants. Tiling microarray probes corresponding to both forward and reverse strands from each window were binned into ∼600 bp windows, and log 2 fold-changes of mutant versus wild-type from duplicate arrays for each mutant strain in each window were averaged. Data smoothing was performed using a three-consecutive-probe window moving average. DOI: http://dx.doi.org/10.7554/eLife.04506.014
Figure Legend Snippet: Derepression of ste11 in mutants deficient in both atf1 and set1 . Expression changes on forward and reverse strands at the ste11 locus in atf1Δ (blue dashed lines) , set1Δ (red solid lines), and atf1Δ set1Δ (dotted purple lines) mutants. Tiling microarray probes corresponding to both forward and reverse strands from each window were binned into ∼600 bp windows, and log 2 fold-changes of mutant versus wild-type from duplicate arrays for each mutant strain in each window were averaged. Data smoothing was performed using a three-consecutive-probe window moving average. DOI: http://dx.doi.org/10.7554/eLife.04506.014

Techniques Used: Expressing, Microarray, Mutagenesis

Atf1 acts as a transcriptional repressor. ( A and B ) Distributions of Atf1 and Set1 at ( A ) fbp1 and ( B ) srk1 (upper panels). Increased H3K4me3 levels at fbp1 and srk1 in atf1 Δ cells (lower panels). Enrichment of Set1, Atf1, and H3K4me3 was determined by chromatin immunoprecipitation (ChIP)–chip. DOI: http://dx.doi.org/10.7554/eLife.04506.013
Figure Legend Snippet: Atf1 acts as a transcriptional repressor. ( A and B ) Distributions of Atf1 and Set1 at ( A ) fbp1 and ( B ) srk1 (upper panels). Increased H3K4me3 levels at fbp1 and srk1 in atf1 Δ cells (lower panels). Enrichment of Set1, Atf1, and H3K4me3 was determined by chromatin immunoprecipitation (ChIP)–chip. DOI: http://dx.doi.org/10.7554/eLife.04506.013

Techniques Used: Chromatin Immunoprecipitation

Colocalization of Set1 and Atf1 at centromeres I and III. Colocalization of Atf1 and Set1 (upper panels) at centromeres I and III (upper panels). Reduced H3K4me3 levels at centromere central cores in atf1 Δ cells (lower panels). Enrichment of Set1, Atf1, and H3K4me3 was analyzed by chromatin immunoprecipitation (ChIP)–chip. DOI: http://dx.doi.org/10.7554/eLife.04506.011
Figure Legend Snippet: Colocalization of Set1 and Atf1 at centromeres I and III. Colocalization of Atf1 and Set1 (upper panels) at centromeres I and III (upper panels). Reduced H3K4me3 levels at centromere central cores in atf1 Δ cells (lower panels). Enrichment of Set1, Atf1, and H3K4me3 was analyzed by chromatin immunoprecipitation (ChIP)–chip. DOI: http://dx.doi.org/10.7554/eLife.04506.011

Techniques Used: Chromatin Immunoprecipitation

Enrichment of Atf1 at repressed loci. Confirmation of Atf1 binding at the rDNA array, ste11 , and pericentromeric heterochromatin ( dg ) was carried out by chromatin immunoprecipitation (ChIP) followed by qPCR. ChIP fold enrichment was calculated relative to input after normalization by primers corresponding to the act1 promoter. (SD, error bars; n = 3 triplicates.) DOI: http://dx.doi.org/10.7554/eLife.04506.012
Figure Legend Snippet: Enrichment of Atf1 at repressed loci. Confirmation of Atf1 binding at the rDNA array, ste11 , and pericentromeric heterochromatin ( dg ) was carried out by chromatin immunoprecipitation (ChIP) followed by qPCR. ChIP fold enrichment was calculated relative to input after normalization by primers corresponding to the act1 promoter. (SD, error bars; n = 3 triplicates.) DOI: http://dx.doi.org/10.7554/eLife.04506.012

Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

27) Product Images from "Cyclin K regulates prereplicative complex assembly to promote mammalian cell proliferation"

Article Title: Cyclin K regulates prereplicative complex assembly to promote mammalian cell proliferation

Journal: Nature Communications

doi: 10.1038/s41467-018-04258-w

Cyclin K expression positively correlates with proliferation. a Analyses of cyclin K protein expression during murine brain development by immunoblotting. Cyclin K protein expression in embryonic (E) and postnatal (P) murine brains correlated with that of Sox2, a marker of neural progenitor cell proliferation. b Analyses of cyclin K protein expression by immunoblotting during murine liver development. c Cyclin K expression detected by immunochemistry during the process of murine liver regeneration in vivo. 2nd, immunochemistry using secondary antibodies alone. 0 h denotes samples collected immediately after partial hepatectomy. Scale bar, 40 μm. d Comparison of cyclin K by immunoblotting in normal and H1299 cancer cells using equal cell numbers as loading control. HFF, neonatal human foreskin fibroblast. e Cyclin K expression detected by immunochemistry in normal and H1299 cancer cells. HFF, neonatal human foreskin fibroblast. Scale bar, 40 μm. f Time course analyses of cyclin K expression by immunoblotting in HCT116 cells treated with protein synthesis inhibitor cycloheximide (CHX, 50 μg/ml). g Time course analyses of cyclin K expression by immunoblotting in cells treated with proteasome inhibitor MG132 (5 μM) in human normal and HCT116 cancer cells. HFF, neonatal human foreskin fibroblast. Experiments were repeated for three times ( a – c ), and more than three times when cell lines were used ( d – g ). Representative results are shown
Figure Legend Snippet: Cyclin K expression positively correlates with proliferation. a Analyses of cyclin K protein expression during murine brain development by immunoblotting. Cyclin K protein expression in embryonic (E) and postnatal (P) murine brains correlated with that of Sox2, a marker of neural progenitor cell proliferation. b Analyses of cyclin K protein expression by immunoblotting during murine liver development. c Cyclin K expression detected by immunochemistry during the process of murine liver regeneration in vivo. 2nd, immunochemistry using secondary antibodies alone. 0 h denotes samples collected immediately after partial hepatectomy. Scale bar, 40 μm. d Comparison of cyclin K by immunoblotting in normal and H1299 cancer cells using equal cell numbers as loading control. HFF, neonatal human foreskin fibroblast. e Cyclin K expression detected by immunochemistry in normal and H1299 cancer cells. HFF, neonatal human foreskin fibroblast. Scale bar, 40 μm. f Time course analyses of cyclin K expression by immunoblotting in HCT116 cells treated with protein synthesis inhibitor cycloheximide (CHX, 50 μg/ml). g Time course analyses of cyclin K expression by immunoblotting in cells treated with proteasome inhibitor MG132 (5 μM) in human normal and HCT116 cancer cells. HFF, neonatal human foreskin fibroblast. Experiments were repeated for three times ( a – c ), and more than three times when cell lines were used ( d – g ). Representative results are shown

Techniques Used: Expressing, Marker, In Vivo

28) Product Images from "Super-Enhancers Promote Transcriptional Dysregulation in Nasopharyngeal Carcinoma"

Article Title: Super-Enhancers Promote Transcriptional Dysregulation in Nasopharyngeal Carcinoma

Journal: Cancer research

doi: 10.1158/0008-5472.CAN-17-1143

Profiling of the SE landscapes in NPC cell lines. A, Hockey stick plots in NPC cells showing input-normalized, rank-ordered H3K27ac signals, highlighting a number of SE-associated genes. B, Heatmaps of ChIP-Seq signal intensity of H3K27ac [±2-kb windows around the center of transcription start site (TSS)] for three NPC cell lines, ordered by their mean signal. C, ChIP-seq profiles of H3K27ac, H3K4me1, H3K4me3, ATAC, and RNA Pol II binding at representative SE (EGFR and MALAT1)- or TE (SKP1)-associated gene loci in C666-1 NPC cells. D, Heatmaps of ChIP-seq profiles of H3K27ac, H3K4me1, H3K4me3, ATAC, and RNA Pol II, ordered by H3K27ac signal (C666-1). E, GO enrichment analysis of SE-associated genes.
Figure Legend Snippet: Profiling of the SE landscapes in NPC cell lines. A, Hockey stick plots in NPC cells showing input-normalized, rank-ordered H3K27ac signals, highlighting a number of SE-associated genes. B, Heatmaps of ChIP-Seq signal intensity of H3K27ac [±2-kb windows around the center of transcription start site (TSS)] for three NPC cell lines, ordered by their mean signal. C, ChIP-seq profiles of H3K27ac, H3K4me1, H3K4me3, ATAC, and RNA Pol II binding at representative SE (EGFR and MALAT1)- or TE (SKP1)-associated gene loci in C666-1 NPC cells. D, Heatmaps of ChIP-seq profiles of H3K27ac, H3K4me1, H3K4me3, ATAC, and RNA Pol II, ordered by H3K27ac signal (C666-1). E, GO enrichment analysis of SE-associated genes.

Techniques Used: Chromatin Immunoprecipitation, Binding Assay

29) Product Images from "Presenilins Regulate Neurotrypsin Gene Expression and Neurotrypsin-dependent Agrin Cleavage via Cyclic AMP Response Element-binding Protein (CREB) Modulation *"

Article Title: Presenilins Regulate Neurotrypsin Gene Expression and Neurotrypsin-dependent Agrin Cleavage via Cyclic AMP Response Element-binding Protein (CREB) Modulation *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M113.513705

PSEN1/2 regulate the transcriptional and epigenetic states of the neurotrypsin promoter. A , top , scheme of neurotrypsin regions tested by ChIP. A–K indicate genomic regions utilized to detect binding of proteins tested. Bottom panels , ChIP analysis of CREB, Sp1, MeCP2, RNA Pol II, CBP, H3K9ac, H3K27ac, H3K9me3, H3K27me3, and H3K4me3 in wild-type ( blue line ), Psen1 / 2 dKO ( red line ), and Psen1 / 2 dKO + h PSEN1 / 2 ( green line ) cells at the neurotrypsin locus. B , left panel , RT-qPCR analysis of Tbp -normalized neurotrypsin mRNA levels in Psen1 / 2 dKO (+h PSEN1 / 2 ) cells after control ( siCTL ) or Mecp2 -specific ( siMeCP2 ) siRNA-mediated knockdown. Right panel , Western blot analysis of MeCP2 and tubulin in the same cells as in the left panel . This experiment was done in triplicate ( n = 3). Mean ± S.E. is shown. p values are shown when significant (Student's t test): ***, p ≤ 0.001. Error bars represent S.E.
Figure Legend Snippet: PSEN1/2 regulate the transcriptional and epigenetic states of the neurotrypsin promoter. A , top , scheme of neurotrypsin regions tested by ChIP. A–K indicate genomic regions utilized to detect binding of proteins tested. Bottom panels , ChIP analysis of CREB, Sp1, MeCP2, RNA Pol II, CBP, H3K9ac, H3K27ac, H3K9me3, H3K27me3, and H3K4me3 in wild-type ( blue line ), Psen1 / 2 dKO ( red line ), and Psen1 / 2 dKO + h PSEN1 / 2 ( green line ) cells at the neurotrypsin locus. B , left panel , RT-qPCR analysis of Tbp -normalized neurotrypsin mRNA levels in Psen1 / 2 dKO (+h PSEN1 / 2 ) cells after control ( siCTL ) or Mecp2 -specific ( siMeCP2 ) siRNA-mediated knockdown. Right panel , Western blot analysis of MeCP2 and tubulin in the same cells as in the left panel . This experiment was done in triplicate ( n = 3). Mean ± S.E. is shown. p values are shown when significant (Student's t test): ***, p ≤ 0.001. Error bars represent S.E.

Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Quantitative RT-PCR, Western Blot

Model for presenilin-dependent regulation of neurotrypsin expression and activity. Wild-type MEFs expressing endogenous PSEN1/2 maintain basal levels of neurotrypsin and neurotrypsin-dependent proteolytic activity ( left panel ). Under these conditions, Sp1 is recruited to the neurotrypsin promoter ( left panel ). The simultaneous presence of repressive H3K27me3 and activating H3K4me3 defines neurotrypsin as a bivalent promoter ( left panel ). Upon presenilin removal, CREB/CBP/RNA Pol II join Sp1 at the neurotrypsin promoter, and repressive H3K27me3 is replaced by the activating H3K9/27ac marks ( middle panel ). These epigenetic changes promote neurotrypsin expression and activity in these cells as shown by increased extracellular accumulation of the neurotrypsin-dependent generated agrin-90 and agrin-22 fragments ( middle panel ). GSK3 activity is required for this regulatory shift and to maintain high levels of neurotrypsin expression and activity in Psen 1/2 dKO cells ( middle panel ). Previous reports showing that presenilins inhibit GSK3 activity and that GSK3 can phosphorylate and activate CREB together with our own data suggest that GSK3 might bridge the presenilins and regulation of neurotrypsin expression via CREB modulation in Psen1/2 dKO cells ( middle panel ). When Psen1 / 2 dKO cells are rescued with h PSEN 1/2, neurotrypsin expression and activity return to basal levels via acquisition of a new repressive epigenetic state at the neurotrypsin promoter such that MeCP2 and the repressive H3K9me3 mark are recruited to the neurotrypsin promoter ( right panel ).
Figure Legend Snippet: Model for presenilin-dependent regulation of neurotrypsin expression and activity. Wild-type MEFs expressing endogenous PSEN1/2 maintain basal levels of neurotrypsin and neurotrypsin-dependent proteolytic activity ( left panel ). Under these conditions, Sp1 is recruited to the neurotrypsin promoter ( left panel ). The simultaneous presence of repressive H3K27me3 and activating H3K4me3 defines neurotrypsin as a bivalent promoter ( left panel ). Upon presenilin removal, CREB/CBP/RNA Pol II join Sp1 at the neurotrypsin promoter, and repressive H3K27me3 is replaced by the activating H3K9/27ac marks ( middle panel ). These epigenetic changes promote neurotrypsin expression and activity in these cells as shown by increased extracellular accumulation of the neurotrypsin-dependent generated agrin-90 and agrin-22 fragments ( middle panel ). GSK3 activity is required for this regulatory shift and to maintain high levels of neurotrypsin expression and activity in Psen 1/2 dKO cells ( middle panel ). Previous reports showing that presenilins inhibit GSK3 activity and that GSK3 can phosphorylate and activate CREB together with our own data suggest that GSK3 might bridge the presenilins and regulation of neurotrypsin expression via CREB modulation in Psen1/2 dKO cells ( middle panel ). When Psen1 / 2 dKO cells are rescued with h PSEN 1/2, neurotrypsin expression and activity return to basal levels via acquisition of a new repressive epigenetic state at the neurotrypsin promoter such that MeCP2 and the repressive H3K9me3 mark are recruited to the neurotrypsin promoter ( right panel ).

Techniques Used: Expressing, Activity Assay, Generated

30) Product Images from "Essential role of the TFIID subunit TAF4 in murine embryogenesis and embryonic stem cell differentiation"

Article Title: Essential role of the TFIID subunit TAF4 in murine embryogenesis and embryonic stem cell differentiation

Journal: Nature Communications

doi: 10.1038/ncomms11063

Abnormal patterning of Taf4a −/− embryos. ( a – o ) (upper row). In situ hybridization with the indicated probes for anterior visceral endoderm (AVE) and mesoderm markers on E7.5 WT embryos. ( b – p ) (lower row). In situ hybridization with the indicated probes on E7.5 Taf4 −/− embryos. ( q – ao ). In situ hybridization with neuronal markers in E9.5 WT or Taf4 −/− embryos (lateral views ( q – ak ); ventral view R dorsal views T-AN) and (lateral views RP-AL, higher magnifications of same embryos s – am ; ventral view U; dorsal views z – ao ). Scale bar, 200 μm in all panels.
Figure Legend Snippet: Abnormal patterning of Taf4a −/− embryos. ( a – o ) (upper row). In situ hybridization with the indicated probes for anterior visceral endoderm (AVE) and mesoderm markers on E7.5 WT embryos. ( b – p ) (lower row). In situ hybridization with the indicated probes on E7.5 Taf4 −/− embryos. ( q – ao ). In situ hybridization with neuronal markers in E9.5 WT or Taf4 −/− embryos (lateral views ( q – ak ); ventral view R dorsal views T-AN) and (lateral views RP-AL, higher magnifications of same embryos s – am ; ventral view U; dorsal views z – ao ). Scale bar, 200 μm in all panels.

Techniques Used: In Situ Hybridization

Taf4 is required for cardiomyocyte differentiation. ( a ) Phase-contrast microscopy and Tnnt2 labelling of EBs differentiated into cardiomyocytes 9 days after plating. ( b ) Phase-contrast microscopy and Tnnt2 labelling of ES cells engineered to re-express exogenous Taf4 differentiated into cardiomyocytes 9 days after plating. All images were taken at × 20 magnification. ( c ) Global comparison of RNA-seq data in Taf4a −/− compared with WT at the EB8 stage. The expression of several cardiomyocyte markers and pluripotency genes are highlighted. ( d ) Comparison of the changes in expression of selected marker genes measured by RNA-seq and by RR–qPCR. Scale bars 50 μm.
Figure Legend Snippet: Taf4 is required for cardiomyocyte differentiation. ( a ) Phase-contrast microscopy and Tnnt2 labelling of EBs differentiated into cardiomyocytes 9 days after plating. ( b ) Phase-contrast microscopy and Tnnt2 labelling of ES cells engineered to re-express exogenous Taf4 differentiated into cardiomyocytes 9 days after plating. All images were taken at × 20 magnification. ( c ) Global comparison of RNA-seq data in Taf4a −/− compared with WT at the EB8 stage. The expression of several cardiomyocyte markers and pluripotency genes are highlighted. ( d ) Comparison of the changes in expression of selected marker genes measured by RNA-seq and by RR–qPCR. Scale bars 50 μm.

Techniques Used: Microscopy, RNA Sequencing Assay, Expressing, Marker, Real-time Polymerase Chain Reaction

Taf4 is required for correct positioning of head and heart. ( a , b ). Schematic representations illustrate normal development at E8.5 and E9.5 highlighting positions of head and heart during embryo turning. ( c – f ) WT and mutant embryos at E9.5 before and after dissection from the yolk sac highlighting the blood-filled yolk sac vascular plexus in control embryos (red arrow in c ) and the embryo protruding partially out of the yolk sac (green arrow in e ). WT embryos show a strong accumulation of blood in the atrium of the heart (red arrow in d ), which was absent in Taf4a −/− embryos ( f ). ( g – i ) Schematic illustration of observed mutant phenotypes at E9.5. All mutant embryos fail to undergo turning at E9.5 and fusion of the allantois to the chorion. In addition to this, mutant embryos develop heart structures either in the exocoelomic cavity ( h – i ) or outside of the visceral yolk sac ( k – l ). ( m – w ) In situ hybridization of wild-type and Taf4a −/− embryos for the cardiac transcription factors Nkx2-5 at E8.5 ( m – p ) or Tbx5 at E8.5 ( q – t ) and E9.5 ( u – w ). ep, ectoplacental cone; all, allantois; ht, heart; flp, forelimb precursors; ift, inflow tract. Scale bar, 500 μm ( c – f ), 300 μm ( h - i ), 200 μm ( m – w ).
Figure Legend Snippet: Taf4 is required for correct positioning of head and heart. ( a , b ). Schematic representations illustrate normal development at E8.5 and E9.5 highlighting positions of head and heart during embryo turning. ( c – f ) WT and mutant embryos at E9.5 before and after dissection from the yolk sac highlighting the blood-filled yolk sac vascular plexus in control embryos (red arrow in c ) and the embryo protruding partially out of the yolk sac (green arrow in e ). WT embryos show a strong accumulation of blood in the atrium of the heart (red arrow in d ), which was absent in Taf4a −/− embryos ( f ). ( g – i ) Schematic illustration of observed mutant phenotypes at E9.5. All mutant embryos fail to undergo turning at E9.5 and fusion of the allantois to the chorion. In addition to this, mutant embryos develop heart structures either in the exocoelomic cavity ( h – i ) or outside of the visceral yolk sac ( k – l ). ( m – w ) In situ hybridization of wild-type and Taf4a −/− embryos for the cardiac transcription factors Nkx2-5 at E8.5 ( m – p ) or Tbx5 at E8.5 ( q – t ) and E9.5 ( u – w ). ep, ectoplacental cone; all, allantois; ht, heart; flp, forelimb precursors; ift, inflow tract. Scale bar, 500 μm ( c – f ), 300 μm ( h - i ), 200 μm ( m – w ).

Techniques Used: Mutagenesis, Dissection, In Situ Hybridization

Neural crest cell markers and primordial germ cells in Taf4 mutant embryos. ( a – o ) In situ hybridizations for neural crest markers Sox10 , Crabp1 and Wnt1 in E9.5 control (lateral views a , f , k dorsal views d , i , n ) and Taf4a −/− embryos (lateral views b , g , l higher magnifications of the same embryos c , h , m dorsal views E, J, O ). ( p – u ). In situ hybridizations at E9.5 with Pou5f1 ( Oct4 ) as a marker for primordial germ cells (PGCs). ba1, first branchial arch; ba2, second branchial arch; ov, otic vesicle; cncc, cranial neural crest cells; mhb, midbrain–hindbrain boundary; nt, neural tube. Scale bar, 200 μm in all panels.
Figure Legend Snippet: Neural crest cell markers and primordial germ cells in Taf4 mutant embryos. ( a – o ) In situ hybridizations for neural crest markers Sox10 , Crabp1 and Wnt1 in E9.5 control (lateral views a , f , k dorsal views d , i , n ) and Taf4a −/− embryos (lateral views b , g , l higher magnifications of the same embryos c , h , m dorsal views E, J, O ). ( p – u ). In situ hybridizations at E9.5 with Pou5f1 ( Oct4 ) as a marker for primordial germ cells (PGCs). ba1, first branchial arch; ba2, second branchial arch; ov, otic vesicle; cncc, cranial neural crest cells; mhb, midbrain–hindbrain boundary; nt, neural tube. Scale bar, 200 μm in all panels.

Techniques Used: Mutagenesis, In Situ, Marker

Taf4 is required for RA-driven neuronal ES cell differentiation. ( a ) Phase-contrast microscopy of RA-treated EBs differentiated into neurons 2 days after plating. ( b ) Tubb3 labelling of RA-treated EBs differentiated into neurons 2 days after plating. ( c , d ). Immunoblots on extracts from differenting ES cells and EBs with the indicated antibodies. The membranes were stained with Ponceau as loading controls. ( e ). Immunoblots on extracts from ES cell lines engineered to re-express recombinant Taf4. ( f ). Phase-contrast microscopy and Tubb3 labelling of RA-treated EBs differentiated into neurons 2 days after plating. All images were taken at × 20 magnification. Scale bars 100 μm.
Figure Legend Snippet: Taf4 is required for RA-driven neuronal ES cell differentiation. ( a ) Phase-contrast microscopy of RA-treated EBs differentiated into neurons 2 days after plating. ( b ) Tubb3 labelling of RA-treated EBs differentiated into neurons 2 days after plating. ( c , d ). Immunoblots on extracts from differenting ES cells and EBs with the indicated antibodies. The membranes were stained with Ponceau as loading controls. ( e ). Immunoblots on extracts from ES cell lines engineered to re-express recombinant Taf4. ( f ). Phase-contrast microscopy and Tubb3 labelling of RA-treated EBs differentiated into neurons 2 days after plating. All images were taken at × 20 magnification. Scale bars 100 μm.

Techniques Used: Cell Differentiation, Microscopy, Western Blot, Staining, Recombinant

Taf4 is required during mid-gestation. ( a – c ) E6.5 stage embryos of the indicated genotypes. ( d – g ). E7.5-stage embryos of the indicated genotypes, g shows a blow up of f . Scale bar, 100 μm. ( h – k ). E8.5-stage embryos of the indicated genotypes, k shows a blow up of j . ( l – o ) E9.5-stage embryos of the indicated genotypes, o shows a blow up of n . Scale bar, 200 μm. ( p – u ) Haematoxylin and eosin-stained sections through WT or mutant embryos at the indicated stages. Scale bar, 100 μm. ep, ectoplacental cone; ve, visceral endoderm; ee, embryonic ectoderm; me, mesoderm;, epc, ectoplacental cavity; ch, chorion; ex, exocoelomic cavity; am, amnion; ac, amniotic cavity; hf, headfold; ht, heart; fg, foregut; hg, hindgut; so, somites; vys, visceral yolk sac; ps, primitive streak.
Figure Legend Snippet: Taf4 is required during mid-gestation. ( a – c ) E6.5 stage embryos of the indicated genotypes. ( d – g ). E7.5-stage embryos of the indicated genotypes, g shows a blow up of f . Scale bar, 100 μm. ( h – k ). E8.5-stage embryos of the indicated genotypes, k shows a blow up of j . ( l – o ) E9.5-stage embryos of the indicated genotypes, o shows a blow up of n . Scale bar, 200 μm. ( p – u ) Haematoxylin and eosin-stained sections through WT or mutant embryos at the indicated stages. Scale bar, 100 μm. ep, ectoplacental cone; ve, visceral endoderm; ee, embryonic ectoderm; me, mesoderm;, epc, ectoplacental cavity; ch, chorion; ex, exocoelomic cavity; am, amnion; ac, amniotic cavity; hf, headfold; ht, heart; fg, foregut; hg, hindgut; so, somites; vys, visceral yolk sac; ps, primitive streak.

Techniques Used: Staining, Mutagenesis

Taf4 is required for activation of neurogenic genes in differentiating ES cells. ( a ). Gene expression changes at different stages of differentiation in Taf4a −/− compared with WT ES cells. The numbers of genes with altered expression and their fold changes at each stage are indicated. ( b ). Global comparison of RNA-seq data in Taf4a −/− compared to WT at the EB8 stage. The expression of several neurogenic and pluripotency genes are highlighted. ( c ). Comparison of the changes in expression of selected marker genes measured by RNA-seq and by RT–qPCR. ( d ). Kinetics of gene expression and their alterations during differentiation are represented as a clustered heatmap. The ontology terms associated with the genes in clusters 2–4 that are most affected by Taf4 loss are indicated. ( e ). Metaprofiles for the indicated ChIP-seq data sets corresponding to genes in clusters 2–4 are shown. For TFIIB, H3K4me3 and H3K27ac the profiles are centred on the TSS. For Pol II the profiles are show over the gene from the transcription start site (TSS) to the transcription termination site (TTS).
Figure Legend Snippet: Taf4 is required for activation of neurogenic genes in differentiating ES cells. ( a ). Gene expression changes at different stages of differentiation in Taf4a −/− compared with WT ES cells. The numbers of genes with altered expression and their fold changes at each stage are indicated. ( b ). Global comparison of RNA-seq data in Taf4a −/− compared to WT at the EB8 stage. The expression of several neurogenic and pluripotency genes are highlighted. ( c ). Comparison of the changes in expression of selected marker genes measured by RNA-seq and by RT–qPCR. ( d ). Kinetics of gene expression and their alterations during differentiation are represented as a clustered heatmap. The ontology terms associated with the genes in clusters 2–4 that are most affected by Taf4 loss are indicated. ( e ). Metaprofiles for the indicated ChIP-seq data sets corresponding to genes in clusters 2–4 are shown. For TFIIB, H3K4me3 and H3K27ac the profiles are centred on the TSS. For Pol II the profiles are show over the gene from the transcription start site (TSS) to the transcription termination site (TTS).

Techniques Used: Activation Assay, Expressing, RNA Sequencing Assay, Marker, Quantitative RT-PCR, Chromatin Immunoprecipitation

31) Product Images from "RNA Polymerase II Stalling Promotes Nucleosome Occlusion and pTEFb Recruitment to Drive Immortalization by Epstein-Barr Virus"

Article Title: RNA Polymerase II Stalling Promotes Nucleosome Occlusion and pTEFb Recruitment to Drive Immortalization by Epstein-Barr Virus

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1002334

DSIF and NELF are recruited to Cp in Mutu III cells. Results show the mean +/− standard deviation of a minimum of three independent experiments carried out using at least 2 chromatin batches. Mutu I signals (open bars) are compared to Mutu III signals (black bars). ChIP using anti-Spt5 antibodies to detect DSIF (A) or anti-NELF-A antibodies to detect NELF (B). Blue-edged graphs show zoomed-in sections to allow better visualization of downstream primer signals.
Figure Legend Snippet: DSIF and NELF are recruited to Cp in Mutu III cells. Results show the mean +/− standard deviation of a minimum of three independent experiments carried out using at least 2 chromatin batches. Mutu I signals (open bars) are compared to Mutu III signals (black bars). ChIP using anti-Spt5 antibodies to detect DSIF (A) or anti-NELF-A antibodies to detect NELF (B). Blue-edged graphs show zoomed-in sections to allow better visualization of downstream primer signals.

Techniques Used: Standard Deviation, Chromatin Immunoprecipitation

32) Product Images from "Constitutive Nucleosome Depletion and Ordered Factor Assembly at the GRP78 Promoter Revealed by Single Molecule Footprinting"

Article Title: Constitutive Nucleosome Depletion and Ordered Factor Assembly at the GRP78 Promoter Revealed by Single Molecule Footprinting

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.0020160

GRP78 Activation during ER Stress LD419 cells were harvested at 0, 0.5, 1, 2, 6, and 16 h after induction with 300 nM TG. The nuclei were purified and treated with M.SssI followed by bisulfite genomic sequencing. (A) Relative GRP78 mRNA levels at the different points after TG induction determined by quantitative RT-PCR (normalized to GAPDH). (B) Sequencing data for the various time points. The diagrams on top, drawn to scale, represent the region analyzed (core promoter amplicon, short) and indicate the distribution density of the 37 CpG sites included in this region. The TIS (bent arrow), TATA box (T), and ERSE elements (E1–E3) are marked. Each horizontal line with a string of circles represents the methylation profile for one DNA molecule. White circles indicate unmethylated, and black circles, methylated CpG sites. The modules, as defined by the correlation analysis in Figure 3 , are color coded: blue, TATA box module; yellow, TIS module; bright green, ERSE1 module; dark green, ERSE2 module; olive, ERSE3 module; dark red, CpGs 49–50 and 51–53 modules; pink, nucleosomal module. The modules were colored if the majority of CpGs comprising the module were protected. The nucleosomal module was colored according to the previous nucleosomal patch definition [ 12 ] where more than two consecutive protected CpGs are considered a patch and the occurrence of one unmethylated CpG site does not break the contiguity of the patch. (C) Protection levels of nucleosomal modules (left), TATA and TIS (middle), and ERSEs (right) were calculated (see Materials and Methods ) and are shown at the different time points. (D) ATF6 enrichment at the ERSE region shown by ChIP analyses on LD419 cells harvested at 0, 1, 4, and 16 h after TG stress induction. DNA was quantified by real-time PCR using primers specific for the indicated four regions of the promoter (as in Figure 1 A).
Figure Legend Snippet: GRP78 Activation during ER Stress LD419 cells were harvested at 0, 0.5, 1, 2, 6, and 16 h after induction with 300 nM TG. The nuclei were purified and treated with M.SssI followed by bisulfite genomic sequencing. (A) Relative GRP78 mRNA levels at the different points after TG induction determined by quantitative RT-PCR (normalized to GAPDH). (B) Sequencing data for the various time points. The diagrams on top, drawn to scale, represent the region analyzed (core promoter amplicon, short) and indicate the distribution density of the 37 CpG sites included in this region. The TIS (bent arrow), TATA box (T), and ERSE elements (E1–E3) are marked. Each horizontal line with a string of circles represents the methylation profile for one DNA molecule. White circles indicate unmethylated, and black circles, methylated CpG sites. The modules, as defined by the correlation analysis in Figure 3 , are color coded: blue, TATA box module; yellow, TIS module; bright green, ERSE1 module; dark green, ERSE2 module; olive, ERSE3 module; dark red, CpGs 49–50 and 51–53 modules; pink, nucleosomal module. The modules were colored if the majority of CpGs comprising the module were protected. The nucleosomal module was colored according to the previous nucleosomal patch definition [ 12 ] where more than two consecutive protected CpGs are considered a patch and the occurrence of one unmethylated CpG site does not break the contiguity of the patch. (C) Protection levels of nucleosomal modules (left), TATA and TIS (middle), and ERSEs (right) were calculated (see Materials and Methods ) and are shown at the different time points. (D) ATF6 enrichment at the ERSE region shown by ChIP analyses on LD419 cells harvested at 0, 1, 4, and 16 h after TG stress induction. DNA was quantified by real-time PCR using primers specific for the indicated four regions of the promoter (as in Figure 1 A).

Techniques Used: Activation Assay, Purification, Genomic Sequencing, Quantitative RT-PCR, Sequencing, Amplification, Methylation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

33) Product Images from "p53 Chromatin Epigenetic Domain Organization and p53 Transcription ▿ Transcription ▿ †"

Article Title: p53 Chromatin Epigenetic Domain Organization and p53 Transcription ▿ Transcription ▿ †

Journal:

doi: 10.1128/MCB.00704-08

Association of several regulatory factors with the p53 epigenetic domains. ChIP assays using anti-CTCF, anti-Topo II, anti-HDAC1, anti-Dnmt1, anti-Brg1, and anti-Topo I antibodies were performed to determine the binding sites of these regulatory factors.
Figure Legend Snippet: Association of several regulatory factors with the p53 epigenetic domains. ChIP assays using anti-CTCF, anti-Topo II, anti-HDAC1, anti-Dnmt1, anti-Brg1, and anti-Topo I antibodies were performed to determine the binding sites of these regulatory factors.

Techniques Used: Chromatin Immunoprecipitation, Binding Assay

34) Product Images from "CTCF Interacts with and Recruits the Largest Subunit of RNA Polymerase II to CTCF Target Sites Genome-Wide ▿"

Article Title: CTCF Interacts with and Recruits the Largest Subunit of RNA Polymerase II to CTCF Target Sites Genome-Wide ▿

Journal:

doi: 10.1128/MCB.01993-06

Genome-wide interaction between CTCF and LS Pol II. (A) ChIP-on-ChIP hybridization analysis revealing the simultaneous presence of CTCF and LS-Pol II epitopes genome-wide. DNA samples from the standard ChIP or serial ChIP assays from proliferating and
Figure Legend Snippet: Genome-wide interaction between CTCF and LS Pol II. (A) ChIP-on-ChIP hybridization analysis revealing the simultaneous presence of CTCF and LS-Pol II epitopes genome-wide. DNA samples from the standard ChIP or serial ChIP assays from proliferating and

Techniques Used: Genome Wide, Chromatin Immunoprecipitation, Hybridization

DNA-bound CTCF and the largest subunit of Pol II simultaneously interact genome-wide to a subset of CTCF binding sites.
Figure Legend Snippet: DNA-bound CTCF and the largest subunit of Pol II simultaneously interact genome-wide to a subset of CTCF binding sites.

Techniques Used: Genome Wide, Binding Assay

DNA-bound CTCF and the largest subunit of Pol II simultaneously interact genome-wide to a subset of CTCF binding sites.
Figure Legend Snippet: DNA-bound CTCF and the largest subunit of Pol II simultaneously interact genome-wide to a subset of CTCF binding sites.

Techniques Used: Genome Wide, Binding Assay

35) Product Images from "STAT3 or USF2 Contributes to HIF Target Gene Specificity"

Article Title: STAT3 or USF2 Contributes to HIF Target Gene Specificity

Journal: PLoS ONE

doi: 10.1371/journal.pone.0072358

N-TADs of HIF1α or HIF2α is required for its functional interaction with STAT3 or USF2 to activate HIF1 or HIF2 target gene promoter. A ) Schematic presentation of HIF1αDPA (Double Proline to Alanine), HIF2αDPA and HIF1α/HIF2α hybrid constructs. B ) Western blot analysis of HIF1α, HIF2α, HIF hybrids and STAT3 to monitor the protein expression during CA9/Luc reporter gene assays. C ) Activation of HIF1 target gene reporter, CA9/Luc, by the indicated plasmids. HIS is an empty vector that contains a histidine tag only. D ) Western blot analysis of HIF1α, HIF2α, HIF hybrids and USF2 to monitor the protein expression during PAI1/Luc reporter gene assays. E ) Activation of HIF2 target gene reporter, PAI1/Luc, by the indicated plasmids.
Figure Legend Snippet: N-TADs of HIF1α or HIF2α is required for its functional interaction with STAT3 or USF2 to activate HIF1 or HIF2 target gene promoter. A ) Schematic presentation of HIF1αDPA (Double Proline to Alanine), HIF2αDPA and HIF1α/HIF2α hybrid constructs. B ) Western blot analysis of HIF1α, HIF2α, HIF hybrids and STAT3 to monitor the protein expression during CA9/Luc reporter gene assays. C ) Activation of HIF1 target gene reporter, CA9/Luc, by the indicated plasmids. HIS is an empty vector that contains a histidine tag only. D ) Western blot analysis of HIF1α, HIF2α, HIF hybrids and USF2 to monitor the protein expression during PAI1/Luc reporter gene assays. E ) Activation of HIF2 target gene reporter, PAI1/Luc, by the indicated plasmids.

Techniques Used: Functional Assay, Construct, Western Blot, Expressing, Activation Assay, Plasmid Preparation

USF2 functions alone or with HIF2α to activate endogenous HIF2 target genes in normoxic Hep3B cells. A ) Western blot analysis of Flag-tagged USF2, HIF1αTM and HIF2αTM to monitor the expression of these plasmids in transfected Hep3B cells. The starts * indicate several HIF2α protein bands expressed from the vector. B ) mRNA levels of HIF1 target genes, PGK1 , GLUT1 , LDHA and CA9 , in normoxic Hep3B cells in response to transient transfection of the indicated plasmids. C ) mRNA levels of HIF2 target genes, EPO , PAI1 , OCT4 and PLAC8 , in normoxic Hep3B cells in response to transient transfection of the indicated plasmids.
Figure Legend Snippet: USF2 functions alone or with HIF2α to activate endogenous HIF2 target genes in normoxic Hep3B cells. A ) Western blot analysis of Flag-tagged USF2, HIF1αTM and HIF2αTM to monitor the expression of these plasmids in transfected Hep3B cells. The starts * indicate several HIF2α protein bands expressed from the vector. B ) mRNA levels of HIF1 target genes, PGK1 , GLUT1 , LDHA and CA9 , in normoxic Hep3B cells in response to transient transfection of the indicated plasmids. C ) mRNA levels of HIF2 target genes, EPO , PAI1 , OCT4 and PLAC8 , in normoxic Hep3B cells in response to transient transfection of the indicated plasmids.

Techniques Used: Western Blot, Expressing, Transfection, Plasmid Preparation

STAT3 or USF2 alone or with HIF1α or HIF2α to activate the cloned promoters of HIF1 or HIF2 target genes in 293T cells. A ) Schematic presentation of the promoters of HIF1 target genes PGK1 and CA9 . B ) Schematic presentation of the promoters/enhancers of HIF2 target genes PAI1 and EPO . Predicted STAT3 {TT(N) 4-6 AA} binding sites (black solid boxes), USF binding sites ( CANNTG ) (gray boxes), and HIF binding sites (HBS, ACGTG) (white boxes) are indicated. Previously validated HIF and USF2 binding sites are indicated by bold boxes. C ) Western blot analysis of Flag-tagged STAT3C, USF2, HIF1αTM and HIF2αTM to monitor the expression of these plasmids in reporter gene assays for Figure 1D-E. Anti-beta actin was for loading control of total protein for this figure and others in the study. D ) Fold of induction of CA9/Luc and PGK1/Luc reporters activated by the indicated plasmids. E ) Fold of induction of PAI1/Luc and EPO/Luc reporters activated by the indicated plasmids.
Figure Legend Snippet: STAT3 or USF2 alone or with HIF1α or HIF2α to activate the cloned promoters of HIF1 or HIF2 target genes in 293T cells. A ) Schematic presentation of the promoters of HIF1 target genes PGK1 and CA9 . B ) Schematic presentation of the promoters/enhancers of HIF2 target genes PAI1 and EPO . Predicted STAT3 {TT(N) 4-6 AA} binding sites (black solid boxes), USF binding sites ( CANNTG ) (gray boxes), and HIF binding sites (HBS, ACGTG) (white boxes) are indicated. Previously validated HIF and USF2 binding sites are indicated by bold boxes. C ) Western blot analysis of Flag-tagged STAT3C, USF2, HIF1αTM and HIF2αTM to monitor the expression of these plasmids in reporter gene assays for Figure 1D-E. Anti-beta actin was for loading control of total protein for this figure and others in the study. D ) Fold of induction of CA9/Luc and PGK1/Luc reporters activated by the indicated plasmids. E ) Fold of induction of PAI1/Luc and EPO/Luc reporters activated by the indicated plasmids.

Techniques Used: Clone Assay, Binding Assay, Western Blot, Expressing

STAT3 and USF2 are enriched on the promoters of HIF1 or HIF2 targets respectively. Chromatin immunoprecipitation was performed in chromatin lysates from normoxic RCC4 ( A ) or hypoxic Hep3B cells ( C ). Antibodies against STAT3 or USF2 were used to co-precipitate STAT3- and USF2-associated genomic DNA. Q-PCR was used to detect the promoters of HIF1 target genes, CA9 and PGK1 , the enhancer or promoter of HIF2 target genes, EPO and PAI1 , or the HIF1/HIF2 common target, VEGF . Results were normalized to input samples as % of input. The relative fold binding was calculated by dividing each of the % input values by the % input value of STAT3 associated with CA9 promoter here in Figure 3A. Similar calculations were made for Figure 3C and ChIP/Re-ChIP in Figure 9. B ) The relative expression levels of CA9 , PAI1 and VEGF mRNAs in normoxic RCC4 cells. D ) The fold of induction of HIF1 target genes CA9 and PGK1 , HIF2 target genes PAI1 and EPO in hypoxic Hep3B cells.
Figure Legend Snippet: STAT3 and USF2 are enriched on the promoters of HIF1 or HIF2 targets respectively. Chromatin immunoprecipitation was performed in chromatin lysates from normoxic RCC4 ( A ) or hypoxic Hep3B cells ( C ). Antibodies against STAT3 or USF2 were used to co-precipitate STAT3- and USF2-associated genomic DNA. Q-PCR was used to detect the promoters of HIF1 target genes, CA9 and PGK1 , the enhancer or promoter of HIF2 target genes, EPO and PAI1 , or the HIF1/HIF2 common target, VEGF . Results were normalized to input samples as % of input. The relative fold binding was calculated by dividing each of the % input values by the % input value of STAT3 associated with CA9 promoter here in Figure 3A. Similar calculations were made for Figure 3C and ChIP/Re-ChIP in Figure 9. B ) The relative expression levels of CA9 , PAI1 and VEGF mRNAs in normoxic RCC4 cells. D ) The fold of induction of HIF1 target genes CA9 and PGK1 , HIF2 target genes PAI1 and EPO in hypoxic Hep3B cells.

Techniques Used: Chromatin Immunoprecipitation, Polymerase Chain Reaction, Binding Assay, Expressing

The HIF2α/USF2 physical interaction requires the C-TAD and N-TAD of the HIF2α protein. A ) WB detection of USF2, HIF1α and HIF2α in RCC4 cell lysate (input), in precipitated materials by the protein A/protein G beads and pre-immuno serum (beads) or in precipitated materials by USF2 antibody and protein A/protein G beads (USF2-IP). B ) Schematic presentation of Flag-tagged full-length (HIF2α), N-terminal (HIF2α-N) or C-terminal (HIF2α-C) halves of HIF2αTM or HIF2α deletion of N-TAD (HIF2α∆NTAD), IH (HIF2α∆IH) or C-TAD (HIF2α∆CTAD). C ) Anti-Flag or anti-HA WB detection of Flag-tagged HIF2α or HA-tagged USF2 protein in cell lysates (Lysates) or in anti-Flag beads precipitated materials (IP). The red starts indicated the positions of HIF2αFL, HIF2α-N and HIF2α-C proteins. The Δ labeled band in USF2+HIF2-C lane was consistently observed, likely expressed from downstream ATG of HIF2α cDNA. D ) Anti-Flag or anti-HA WB detection of Flag-tagged HIF2α or HA-tagged USF2 protein in cell lysates (Lysates) or in anti-Flag beads precipitated materials (IP). The background signals in USF2-HA only lane was deducted from the “anti-HA IP’ signal to calculate the % IP.
Figure Legend Snippet: The HIF2α/USF2 physical interaction requires the C-TAD and N-TAD of the HIF2α protein. A ) WB detection of USF2, HIF1α and HIF2α in RCC4 cell lysate (input), in precipitated materials by the protein A/protein G beads and pre-immuno serum (beads) or in precipitated materials by USF2 antibody and protein A/protein G beads (USF2-IP). B ) Schematic presentation of Flag-tagged full-length (HIF2α), N-terminal (HIF2α-N) or C-terminal (HIF2α-C) halves of HIF2αTM or HIF2α deletion of N-TAD (HIF2α∆NTAD), IH (HIF2α∆IH) or C-TAD (HIF2α∆CTAD). C ) Anti-Flag or anti-HA WB detection of Flag-tagged HIF2α or HA-tagged USF2 protein in cell lysates (Lysates) or in anti-Flag beads precipitated materials (IP). The red starts indicated the positions of HIF2αFL, HIF2α-N and HIF2α-C proteins. The Δ labeled band in USF2+HIF2-C lane was consistently observed, likely expressed from downstream ATG of HIF2α cDNA. D ) Anti-Flag or anti-HA WB detection of Flag-tagged HIF2α or HA-tagged USF2 protein in cell lysates (Lysates) or in anti-Flag beads precipitated materials (IP). The background signals in USF2-HA only lane was deducted from the “anti-HA IP’ signal to calculate the % IP.

Techniques Used: Western Blot, Labeling

The role of HIF and USF2 binding sites on HIF target gene specificity. A ) Schematic presentation of the CA9 promoters, a HIF1 target gene. Construct 1 was generated by inserting 2 copies of -191 HBS of PAI1 promoter, a HIF2 target gene near the -13 HBS of CA9 promoter. Constructs 2 and 3 were generated by inserting -684 and -565 USF2 binding sites of PAI1 promoter near the -13 HBS (construct 2) or near -1001 of CA9 promoter (construct 3). B ) Schematic presentation of a shorter version of CA9 promoters (-506/+25). Construct 4 was generated by replacing the original -13 HBS with -191 HBS of PAI1 promoter. Constructs 5 and 6 were generated by replacing the STAT3 binding sites at -499 and -464 of CA9 promoter with -191 HBS (construct 5) or -684 and -565 USF2 binding sites (construct 6) from the PAI1 promoter. Construct 7 was made by replaced -13 HBS and -499 and -464 STAT3 binding sites in the CA9 promoter with HBS and USF2 binding sites from PAI1 promoter. C ) Fold of induction of CA9/Luc reporters (-1096/+25) activated by the indicated plasmids. D ) Fold of induction of CA9/Luc reporters (-506/+25) activated by the indicated plasmids. The same activators used in Figure 1 were used for experiments here.
Figure Legend Snippet: The role of HIF and USF2 binding sites on HIF target gene specificity. A ) Schematic presentation of the CA9 promoters, a HIF1 target gene. Construct 1 was generated by inserting 2 copies of -191 HBS of PAI1 promoter, a HIF2 target gene near the -13 HBS of CA9 promoter. Constructs 2 and 3 were generated by inserting -684 and -565 USF2 binding sites of PAI1 promoter near the -13 HBS (construct 2) or near -1001 of CA9 promoter (construct 3). B ) Schematic presentation of a shorter version of CA9 promoters (-506/+25). Construct 4 was generated by replacing the original -13 HBS with -191 HBS of PAI1 promoter. Constructs 5 and 6 were generated by replacing the STAT3 binding sites at -499 and -464 of CA9 promoter with -191 HBS (construct 5) or -684 and -565 USF2 binding sites (construct 6) from the PAI1 promoter. Construct 7 was made by replaced -13 HBS and -499 and -464 STAT3 binding sites in the CA9 promoter with HBS and USF2 binding sites from PAI1 promoter. C ) Fold of induction of CA9/Luc reporters (-1096/+25) activated by the indicated plasmids. D ) Fold of induction of CA9/Luc reporters (-506/+25) activated by the indicated plasmids. The same activators used in Figure 1 were used for experiments here.

Techniques Used: Binding Assay, Construct, Generated

HIF1 or HIF2 target gene promoters/enhancers are bound by distinct HIF1α/Pol II or HIF2α/Pol II transcriptional complexes and formation of these transcriptional complexes depends on STAT3 or USF2 activity. Sonicated chromatin from normoxic RCC4, RCC4/STAT3 shRNA, RCC4/USF2 shRNA or hypoxic Hep3B cells or Hep3B/USF2 shRNA cell was subjected to anti-Pol II ChIP, the precipitated protein/DNA complexes were then subjected to a secondary ChIP using HIF1α or HIF2α antibodies. Precipitated DNA was analyzed for HIF target gene promoter and results were displayed as percent of relative fold of binding. A ) Detection of Pol II/HIF1α or Pol II/HIF2α complexes on the promoters of HIF1 target genes, CA9 and PGK1 (two left columns) and mRNA levels of CA9 and PGK1 in normoxic RCC4, RCC4/STAT3 shRNA and RCC4/ USF2 shRNA cells (two right columns). B ) Detection of Pol II/HIF1α or Pol II/HIF2α complexes on the promoter or enhancer of HIF2 target genes, PAI1 and EPO (two left columns) and mRNA levels of PAI-1 and EPO in normoxic RCC4, RCC4/STAT3 shRNA and RCC4/ USF2 shRNA cells (two right columns). C ) Detection of Pol II/HIF1α or Pol II/HIF2α complexes on the promoters of HIF1 target genes, CA9 and PGK1 and fold of induction of CA9 and PGK1 gene expression in hypoxic Hep3B and Hep3B/USF2 shRNA cells. D ) Detection of Pol II/HIF1α or Pol II/HIF2α complexes on the promoter or enhancer of HIF2 target genes, PAI1 and EPO and fold of induction of PAI1 and EPO in hypoxic Hep3B and Hep3B/USF2 shRNA cells.
Figure Legend Snippet: HIF1 or HIF2 target gene promoters/enhancers are bound by distinct HIF1α/Pol II or HIF2α/Pol II transcriptional complexes and formation of these transcriptional complexes depends on STAT3 or USF2 activity. Sonicated chromatin from normoxic RCC4, RCC4/STAT3 shRNA, RCC4/USF2 shRNA or hypoxic Hep3B cells or Hep3B/USF2 shRNA cell was subjected to anti-Pol II ChIP, the precipitated protein/DNA complexes were then subjected to a secondary ChIP using HIF1α or HIF2α antibodies. Precipitated DNA was analyzed for HIF target gene promoter and results were displayed as percent of relative fold of binding. A ) Detection of Pol II/HIF1α or Pol II/HIF2α complexes on the promoters of HIF1 target genes, CA9 and PGK1 (two left columns) and mRNA levels of CA9 and PGK1 in normoxic RCC4, RCC4/STAT3 shRNA and RCC4/ USF2 shRNA cells (two right columns). B ) Detection of Pol II/HIF1α or Pol II/HIF2α complexes on the promoter or enhancer of HIF2 target genes, PAI1 and EPO (two left columns) and mRNA levels of PAI-1 and EPO in normoxic RCC4, RCC4/STAT3 shRNA and RCC4/ USF2 shRNA cells (two right columns). C ) Detection of Pol II/HIF1α or Pol II/HIF2α complexes on the promoters of HIF1 target genes, CA9 and PGK1 and fold of induction of CA9 and PGK1 gene expression in hypoxic Hep3B and Hep3B/USF2 shRNA cells. D ) Detection of Pol II/HIF1α or Pol II/HIF2α complexes on the promoter or enhancer of HIF2 target genes, PAI1 and EPO and fold of induction of PAI1 and EPO in hypoxic Hep3B and Hep3B/USF2 shRNA cells.

Techniques Used: Activity Assay, Sonication, shRNA, Chromatin Immunoprecipitation, Binding Assay, Expressing

Model for HIF target gene specificity. Although HIF1α/ARNT and HIF2α/ARNT lack binding specificity, HIF1α/ARNT and HIF2α/ARNT activate unique target genes. Other transcription factors such as STAT3 and USF2 are required for HIF1 or HIF2 target gene activation, these HIF1 or HIF2 specific trascription partners contribute to HIF target gene specificity by mechanisms including: 1) A specific/preferential binding of STAT3 or USF2 to the promoters of HIF1 or HIF2 target genes. 2) Specific physical and functional interaction of HIF1α with STAT3, or HIF2α with USF2. and 3) HIF1α/ARNT specific transcription partners such as STAT3 or HIF2α/ARNT specific transcription partners such as USF2 are required for the formation of the functional transcription complexes on the promoters of HIF1 or HIF2 target genes respectively.
Figure Legend Snippet: Model for HIF target gene specificity. Although HIF1α/ARNT and HIF2α/ARNT lack binding specificity, HIF1α/ARNT and HIF2α/ARNT activate unique target genes. Other transcription factors such as STAT3 and USF2 are required for HIF1 or HIF2 target gene activation, these HIF1 or HIF2 specific trascription partners contribute to HIF target gene specificity by mechanisms including: 1) A specific/preferential binding of STAT3 or USF2 to the promoters of HIF1 or HIF2 target genes. 2) Specific physical and functional interaction of HIF1α with STAT3, or HIF2α with USF2. and 3) HIF1α/ARNT specific transcription partners such as STAT3 or HIF2α/ARNT specific transcription partners such as USF2 are required for the formation of the functional transcription complexes on the promoters of HIF1 or HIF2 target genes respectively.

Techniques Used: Binding Assay, Activation Assay, Functional Assay

36) Product Images from "Insulin-response epigenetic activation of Egr-1 and JunB genes at the nuclear periphery by A-type lamin-associated pY19-Caveolin-2 in the inner nuclear membrane"

Article Title: Insulin-response epigenetic activation of Egr-1 and JunB genes at the nuclear periphery by A-type lamin-associated pY19-Caveolin-2 in the inner nuclear membrane

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkv181

pY19-Cav-2 in the INM directly interacts with lamin A/C at the nuclear periphery in response to insulin. ( A and B ) Cav-2 interaction with lamin A/C but not lamin B1 in endogenous Cav-2-expressing Hirc-B cells. Insulin-induced interaction between Cav-2 and lamin A/C ( A, a ), pY19-Cav-2 and lamin A/C ( A , b ), or Cav-2 and lamin B1 ( A , c ) at the nuclear periphery was determined by Duolink PLA in Hirc-B cells treated with or without 100 nM insulin for 30 min. The in situ interactions between lamin A/C and emerin ( B , a ) and between lamin B1 and LBR ( B , b ) in Hirc-B cells were visualized by Duolink PLA. White dots in the gray-scale images represent the clusters of protein–protein interactions. Scale bars, 20 μm. ( C ) The binding motif of Cav-2 (residues 47–70) to lamin A/C. Nuclear lysates from HEK293T cells expressing no endogenous Caveolins were subjected to in vitro binding assay using MBP vector, MBP-Cav-2, MBP-Δ1–13-Cav-2, MBP-Δ1–46-Cav-2, MBP-Δ1–70-Cav-2, MBP-Δ47–86-Cav-2 or MBP-Δ120–162-Cav-2. ( D ) Association of Cav-2 in the INM with lamin A/C. HEK293T cells expressed with Cav-2-PAGFP or Δ47–86-Cav-2-PAGFP, or co-transfected with Cav-2-PAGFP or Δ47–86-Cav-2-PAGFP and siLamin A/C were subjected to photoactivation. The graph shows FI of Cav-2-PAGFP or Δ47–86-Cav-2-PAGFP in the NE region before (1: solid box) and after (2: dotted box) photoactivation (mean ± SE, n = 5). Scale bars, 5 μm.
Figure Legend Snippet: pY19-Cav-2 in the INM directly interacts with lamin A/C at the nuclear periphery in response to insulin. ( A and B ) Cav-2 interaction with lamin A/C but not lamin B1 in endogenous Cav-2-expressing Hirc-B cells. Insulin-induced interaction between Cav-2 and lamin A/C ( A, a ), pY19-Cav-2 and lamin A/C ( A , b ), or Cav-2 and lamin B1 ( A , c ) at the nuclear periphery was determined by Duolink PLA in Hirc-B cells treated with or without 100 nM insulin for 30 min. The in situ interactions between lamin A/C and emerin ( B , a ) and between lamin B1 and LBR ( B , b ) in Hirc-B cells were visualized by Duolink PLA. White dots in the gray-scale images represent the clusters of protein–protein interactions. Scale bars, 20 μm. ( C ) The binding motif of Cav-2 (residues 47–70) to lamin A/C. Nuclear lysates from HEK293T cells expressing no endogenous Caveolins were subjected to in vitro binding assay using MBP vector, MBP-Cav-2, MBP-Δ1–13-Cav-2, MBP-Δ1–46-Cav-2, MBP-Δ1–70-Cav-2, MBP-Δ47–86-Cav-2 or MBP-Δ120–162-Cav-2. ( D ) Association of Cav-2 in the INM with lamin A/C. HEK293T cells expressed with Cav-2-PAGFP or Δ47–86-Cav-2-PAGFP, or co-transfected with Cav-2-PAGFP or Δ47–86-Cav-2-PAGFP and siLamin A/C were subjected to photoactivation. The graph shows FI of Cav-2-PAGFP or Δ47–86-Cav-2-PAGFP in the NE region before (1: solid box) and after (2: dotted box) photoactivation (mean ± SE, n = 5). Scale bars, 5 μm.

Techniques Used: Expressing, Proximity Ligation Assay, In Situ, Binding Assay, In Vitro, Plasmid Preparation, Transfection

37) Product Images from "14-3-3 (Bmh) Proteins Regulate Combinatorial Transcription following RNA Polymerase II Recruitment by Binding at Adr1-Dependent Promoters in Saccharomyces cerevisiae"

Article Title: 14-3-3 (Bmh) Proteins Regulate Combinatorial Transcription following RNA Polymerase II Recruitment by Binding at Adr1-Dependent Promoters in Saccharomyces cerevisiae

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.01226-12

GBD-Adr1 binds to GAL genes and recruits RNA Pol II in the presence of Bmh1 activity. (A) GBD ChIP. YLL908 ( BMH1 bmh2 Δ) and YLL1087 ( bmh1-ts bmh2 Δ) were transformed with a plasmid expressing either GBD (pOBD2) or GBD-Adr1 (154–424)
Figure Legend Snippet: GBD-Adr1 binds to GAL genes and recruits RNA Pol II in the presence of Bmh1 activity. (A) GBD ChIP. YLL908 ( BMH1 bmh2 Δ) and YLL1087 ( bmh1-ts bmh2 Δ) were transformed with a plasmid expressing either GBD (pOBD2) or GBD-Adr1 (154–424)

Techniques Used: Activity Assay, Chromatin Immunoprecipitation, Transformation Assay, Plasmid Preparation, Expressing

38) Product Images from "Mammalian E-type Cyclins Control Chromosome Pairing, Telomere Stability and CDK2 Localization in Male Meiosis"

Article Title: Mammalian E-type Cyclins Control Chromosome Pairing, Telomere Stability and CDK2 Localization in Male Meiosis

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1004165

Meiotic sex chromosome inactivation appears unaffected by depletion of E-type cyclins. Chromosome spreads from adult wild type (WT) (a–b), E1+/+E2−/− (c–d), E1+/ΔE2−/− (e–f) and E1Δ/ΔE2−/− spermatocytes (g–h) immunostained for SUMO-1 (red) (a,c,e,g) or RNA pol II (red) (b,d,f,h) and SYCP3 (green). SUMO-1 is in the sex chromosomes (XY) in mid-pachytene to diplotene in WT (a), E1+/+E2−/− (c), and E1+/ΔE2−/− (e) spermatocytes. SUMO-1 also localizes in the telomeres and telomeric and subtelomeric regions of some chromosomes (c,e, white arrows) and in the chromatin of unsynapsed autosomes (e, purple arrow). (g) In most E1Δ/ΔE2−/− spermatocytes, SUMO-1 was absent from the chromatin. In E1+/+E2−/− (d) and E1+/ΔE2−/− (f) pachytene spermatocytes, RNA pol II is distributed throughout almost all the chromatin except where the sex chromosomes (X,Y) are localized, similar to WT (b). In E1Δ/ΔE2−/− testes, RNA pol II was not detected in most of the spermatocytes (h).
Figure Legend Snippet: Meiotic sex chromosome inactivation appears unaffected by depletion of E-type cyclins. Chromosome spreads from adult wild type (WT) (a–b), E1+/+E2−/− (c–d), E1+/ΔE2−/− (e–f) and E1Δ/ΔE2−/− spermatocytes (g–h) immunostained for SUMO-1 (red) (a,c,e,g) or RNA pol II (red) (b,d,f,h) and SYCP3 (green). SUMO-1 is in the sex chromosomes (XY) in mid-pachytene to diplotene in WT (a), E1+/+E2−/− (c), and E1+/ΔE2−/− (e) spermatocytes. SUMO-1 also localizes in the telomeres and telomeric and subtelomeric regions of some chromosomes (c,e, white arrows) and in the chromatin of unsynapsed autosomes (e, purple arrow). (g) In most E1Δ/ΔE2−/− spermatocytes, SUMO-1 was absent from the chromatin. In E1+/+E2−/− (d) and E1+/ΔE2−/− (f) pachytene spermatocytes, RNA pol II is distributed throughout almost all the chromatin except where the sex chromosomes (X,Y) are localized, similar to WT (b). In E1Δ/ΔE2−/− testes, RNA pol II was not detected in most of the spermatocytes (h).

Techniques Used:

39) Product Images from "miR675 Accelerates Malignant Transformation of Mesenchymal Stem Cells by Blocking DNA Mismatch Repair"

Article Title: miR675 Accelerates Malignant Transformation of Mesenchymal Stem Cells by Blocking DNA Mismatch Repair

Journal: Molecular Therapy. Nucleic Acids

doi: 10.1016/j.omtn.2018.11.010

miR675 Affects the Interaction among hMSH6, H3k36me3, and SKP2 in the Human Mesenchymal Stem Cells Infected with rLV and rLV-miR675, Respectively (A) Anti-STED2 co-immunoprecipitation (coIP) followed by western blotting with anti-P62 and anti-histone 3. IgG IP was the negative control. INPUT refers to western blotting with anti-STED2. (B) Anti-SETD2 coIP followed by western blotting with anti-histone 3. IgG IP was the negative control. INPUT refers to western blotting with anti-SETD2. (C) (a) Western blotting with anti-H3K36me1, anti-H3K36me2, and anti-H3K36me1. β-actin was the internal control. (b) The gray scan analysis of positive bands of western blotting. (D) Anti-hMSH6 coIP followed by western blotting with anti-H3K36me3 and anti-SKP2. IgG IP was the negative control. INPUT refers to western blotting with anti-hMSH6. (E) Anti-hMSH6 coIP followed by western blotting with anti-SKP2 in the human mesenchymal stem cells, including the rLV and rLV-miR675 plus pCMV6-AC-GFP-JMJD2A groups. IgG IP was the negative control. INPUT refers to western blotting with anti-hMSH6.
Figure Legend Snippet: miR675 Affects the Interaction among hMSH6, H3k36me3, and SKP2 in the Human Mesenchymal Stem Cells Infected with rLV and rLV-miR675, Respectively (A) Anti-STED2 co-immunoprecipitation (coIP) followed by western blotting with anti-P62 and anti-histone 3. IgG IP was the negative control. INPUT refers to western blotting with anti-STED2. (B) Anti-SETD2 coIP followed by western blotting with anti-histone 3. IgG IP was the negative control. INPUT refers to western blotting with anti-SETD2. (C) (a) Western blotting with anti-H3K36me1, anti-H3K36me2, and anti-H3K36me1. β-actin was the internal control. (b) The gray scan analysis of positive bands of western blotting. (D) Anti-hMSH6 coIP followed by western blotting with anti-H3K36me3 and anti-SKP2. IgG IP was the negative control. INPUT refers to western blotting with anti-hMSH6. (E) Anti-hMSH6 coIP followed by western blotting with anti-SKP2 in the human mesenchymal stem cells, including the rLV and rLV-miR675 plus pCMV6-AC-GFP-JMJD2A groups. IgG IP was the negative control. INPUT refers to western blotting with anti-hMSH6.

Techniques Used: Infection, Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Negative Control

miR675 Delays the hMSH6-H3k36me3-Skp2 Ternary Complex Occupancy on the Mismatch DNA in the Human Mesenchymal Stem Cells Infected with rLV and rLV-miR675, Respectively (A) (a) Super-EMSA (gel shift) with biotin-DNA probe (including mismatch and match DNA double strands) and anti-histone H3.3 antibody. The intensity of the band was examined by western blotting with anti-biotin. (b) The gray scan analysis of positive bands of super-EMSA. (B) Biotin-mismatch probe pull-down followed by western blotting with anti-histone 3.3, anti-histone 3, anti-SKP2, anti-hMSH6, and anti-H3K36me3. Biotin was the INPUT and histone was the internal control. (C) Chromatin immunoprecipitation (ChIP) with anti-histone 3.3, anti-histone H3, anti-SKP2, anti-hMSH6, and anti-H3K36me3 followed by PCR with damaged DNA primers. IgG ChIP was the negative control and damaged DNA was the INPUT. (D) Biotin-match double DNA probe pull-down followed by western blotting with anti-histone H3.3, anti-histone H3, anti-SKP2, anti-hMSH6, and anti-H3K36me3. Biotin was the INPUT and histone was the internal control. (E) ChIP with anti-histone 3.3, anti-histone H3, anti-SKP2, anti-hMSH6, and anti-H3K36me3 followed by PCR with match double DNA primers. IgG ChIP was the negative control and match double DNA was the INPUT.
Figure Legend Snippet: miR675 Delays the hMSH6-H3k36me3-Skp2 Ternary Complex Occupancy on the Mismatch DNA in the Human Mesenchymal Stem Cells Infected with rLV and rLV-miR675, Respectively (A) (a) Super-EMSA (gel shift) with biotin-DNA probe (including mismatch and match DNA double strands) and anti-histone H3.3 antibody. The intensity of the band was examined by western blotting with anti-biotin. (b) The gray scan analysis of positive bands of super-EMSA. (B) Biotin-mismatch probe pull-down followed by western blotting with anti-histone 3.3, anti-histone 3, anti-SKP2, anti-hMSH6, and anti-H3K36me3. Biotin was the INPUT and histone was the internal control. (C) Chromatin immunoprecipitation (ChIP) with anti-histone 3.3, anti-histone H3, anti-SKP2, anti-hMSH6, and anti-H3K36me3 followed by PCR with damaged DNA primers. IgG ChIP was the negative control and damaged DNA was the INPUT. (D) Biotin-match double DNA probe pull-down followed by western blotting with anti-histone H3.3, anti-histone H3, anti-SKP2, anti-hMSH6, and anti-H3K36me3. Biotin was the INPUT and histone was the internal control. (E) ChIP with anti-histone 3.3, anti-histone H3, anti-SKP2, anti-hMSH6, and anti-H3K36me3 followed by PCR with match double DNA primers. IgG ChIP was the negative control and match double DNA was the INPUT.

Techniques Used: Infection, Electrophoretic Mobility Shift Assay, Western Blot, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Negative Control

The Schematic Illustrates a Model of the Differentiation of Human Mesenchymal Stem Cells into Cancer Cells via miR675 Oncogenic miR675 promotes the interaction between CREB and P300, which leads to the high expression of P62. That P62 competes with STED2 to bind histone H3 greatly reduces the STED2-binding capacity with substrate histone H3, triggering a reduction of three methylations on histone H3 36th lysine (H3K36me3); thereby, the H3K36me3-hMSH6-SKP2 tri-complex is decreased. Meanwhile, the ternary complex occupancy capacity on chromosome is absolutely reduced, preventing normal DNA repair. By virtue of the reductive degradation ability of SKP2 for aging histone H3.3 bound to damaged DNA, the aging histone H3.3 repair is delayed and eliminated. That the damaged DNA escaped repair can lead to the abnormal expression of some cell cycle- and metabolism-related genes, causing the human mesenchymal stem cell malignant transformation.
Figure Legend Snippet: The Schematic Illustrates a Model of the Differentiation of Human Mesenchymal Stem Cells into Cancer Cells via miR675 Oncogenic miR675 promotes the interaction between CREB and P300, which leads to the high expression of P62. That P62 competes with STED2 to bind histone H3 greatly reduces the STED2-binding capacity with substrate histone H3, triggering a reduction of three methylations on histone H3 36th lysine (H3K36me3); thereby, the H3K36me3-hMSH6-SKP2 tri-complex is decreased. Meanwhile, the ternary complex occupancy capacity on chromosome is absolutely reduced, preventing normal DNA repair. By virtue of the reductive degradation ability of SKP2 for aging histone H3.3 bound to damaged DNA, the aging histone H3.3 repair is delayed and eliminated. That the damaged DNA escaped repair can lead to the abnormal expression of some cell cycle- and metabolism-related genes, causing the human mesenchymal stem cell malignant transformation.

Techniques Used: Expressing, Binding Assay, Transformation Assay

miR675 Inhibits DNA Damage Repair and Decreases Aging Histone H3.3 Degradation in the Human Mesenchymal Stem Cells Infected with rLV, rLV-miR675, and rLV-miR675 Plus rLV-Cas9-P62, Respectively (A) Restriction endonuclease analysis with BamHI and EcoRI for plasmid DNA injury repair. (B) (a) Anti-SKP2 coIP followed by western blotting with anti-histone 3.3. IgG IP was the negative control. INPUT refers to western blotting with anti-histone H3.3. (b) The gray scan analysis of positive bands. (C) Anti-Ub coIP followed by western blotting with anti-histone H3.3. IgG IP was the negative control. INPUT refers to western blotting with β-actin.
Figure Legend Snippet: miR675 Inhibits DNA Damage Repair and Decreases Aging Histone H3.3 Degradation in the Human Mesenchymal Stem Cells Infected with rLV, rLV-miR675, and rLV-miR675 Plus rLV-Cas9-P62, Respectively (A) Restriction endonuclease analysis with BamHI and EcoRI for plasmid DNA injury repair. (B) (a) Anti-SKP2 coIP followed by western blotting with anti-histone 3.3. IgG IP was the negative control. INPUT refers to western blotting with anti-histone H3.3. (b) The gray scan analysis of positive bands. (C) Anti-Ub coIP followed by western blotting with anti-histone H3.3. IgG IP was the negative control. INPUT refers to western blotting with β-actin.

Techniques Used: Infection, Plasmid Preparation, Co-Immunoprecipitation Assay, Western Blot, Negative Control

40) Product Images from "Intron 1 GATA site enhances ALAS2 expression indispensably during erythroid differentiation"

Article Title: Intron 1 GATA site enhances ALAS2 expression indispensably during erythroid differentiation

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkw901

The intron 1 GATA site physically interacts with the promoter and the intron 8 GATA site to form a long-rang enhancer chromatin loop. ( A ) Schematic of the ALAS2 proximal promoter region and intron 1 and 8 enhancer regions used for ChIP-qPCR. The examined promoter region in this study is located at chromosomal position X: 55057352–55057511, which includes the GATA site; the intron 1 enhancer region is located at chromosomal position X: 55054583–55054697, which includes the GATA site and its adjacent E-box; the intron 8 enhancer region is located at chromosomal position X: 55041577–55041687, which includes the GATA site and its adjacent E-box. The ChIP-qPCR primers for the proximal promoter region, intron 1 enhancer region and intron 8 enhancer region are shown in Supplementary Table S1. ( B–G ) The enrichment of GATA1, TAL1, FOG1, Pol II, LDB1 and LMO2 at the promoter, intron 1 and intron 8 enhancer regions was demonstrated by ChIP-qPCR in K562 cells. Normal rabbit IgG was employed as the control in all ChIP assays. The DNA sequences at the exon 4-intron 4 junction (exon 4/intron 4) acts as a negative control region for all ChIP-qPCR assays. ( H and I ) 3C assays indicate that the intron 8 GATA site physically interacts with the intron 1 GATA site (H) and promoter (I) in K562 cells. Vertical lines represent XbaI restriction sites; arrows indicate PCR primer sites and direction. Anchor symbols mark the anchoring primer. The experiments were performed independently in triplicate. * P
Figure Legend Snippet: The intron 1 GATA site physically interacts with the promoter and the intron 8 GATA site to form a long-rang enhancer chromatin loop. ( A ) Schematic of the ALAS2 proximal promoter region and intron 1 and 8 enhancer regions used for ChIP-qPCR. The examined promoter region in this study is located at chromosomal position X: 55057352–55057511, which includes the GATA site; the intron 1 enhancer region is located at chromosomal position X: 55054583–55054697, which includes the GATA site and its adjacent E-box; the intron 8 enhancer region is located at chromosomal position X: 55041577–55041687, which includes the GATA site and its adjacent E-box. The ChIP-qPCR primers for the proximal promoter region, intron 1 enhancer region and intron 8 enhancer region are shown in Supplementary Table S1. ( B–G ) The enrichment of GATA1, TAL1, FOG1, Pol II, LDB1 and LMO2 at the promoter, intron 1 and intron 8 enhancer regions was demonstrated by ChIP-qPCR in K562 cells. Normal rabbit IgG was employed as the control in all ChIP assays. The DNA sequences at the exon 4-intron 4 junction (exon 4/intron 4) acts as a negative control region for all ChIP-qPCR assays. ( H and I ) 3C assays indicate that the intron 8 GATA site physically interacts with the intron 1 GATA site (H) and promoter (I) in K562 cells. Vertical lines represent XbaI restriction sites; arrows indicate PCR primer sites and direction. Anchor symbols mark the anchoring primer. The experiments were performed independently in triplicate. * P

Techniques Used: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Negative Control, Polymerase Chain Reaction

Deletion of the int-1-GATA site abolishes the enhancer loop and almost entirely removes Pol II enrichment at the intron 1 enhancer region and promoter. ( A and B ) The formation of a chromatin loop between the intron 8 GATA site and the intron 1 GATA site (A) or the promoter region (B) in int1Δ6, int8Δ4 and WT K562 cells was measured by 3C assays. ( C–F ) The binding of TAL1, LDB1, LMO2 and Pol II to the promoter and the intron 1 and 8 enhancer regions was measured by ChIP-qPCR in int1Δ6, int8Δ4 and WT K562 cells, respectively. ( G ) The occupancy of Gata1 at the intron 1 GATA site in yolk sac cells of Alas2 X/X , Alas2 X/Y , Alas2 Δ13/X , Alas2 Δ13/Y mice at E11.5 by ChIP-qPCR. ( H ) The occupancy of Pol II at the intron 1 GATA site and promoter in yolk sac cells of Alas2 X/X , Alas2 X/Y , Alas2 Δ13/X , Alas2 Δ13/Y mice at E11.5 by ChIP-qPCR. Normal rabbit IgG was employed as the control in all ChIP assays. The DNA sequence at the exon 4-intron 4 junction (exon 4/intron 4) acts as a negative control region for all ChIP-qPCR assays. All experiments were performed independently in triplicate. * P
Figure Legend Snippet: Deletion of the int-1-GATA site abolishes the enhancer loop and almost entirely removes Pol II enrichment at the intron 1 enhancer region and promoter. ( A and B ) The formation of a chromatin loop between the intron 8 GATA site and the intron 1 GATA site (A) or the promoter region (B) in int1Δ6, int8Δ4 and WT K562 cells was measured by 3C assays. ( C–F ) The binding of TAL1, LDB1, LMO2 and Pol II to the promoter and the intron 1 and 8 enhancer regions was measured by ChIP-qPCR in int1Δ6, int8Δ4 and WT K562 cells, respectively. ( G ) The occupancy of Gata1 at the intron 1 GATA site in yolk sac cells of Alas2 X/X , Alas2 X/Y , Alas2 Δ13/X , Alas2 Δ13/Y mice at E11.5 by ChIP-qPCR. ( H ) The occupancy of Pol II at the intron 1 GATA site and promoter in yolk sac cells of Alas2 X/X , Alas2 X/Y , Alas2 Δ13/X , Alas2 Δ13/Y mice at E11.5 by ChIP-qPCR. Normal rabbit IgG was employed as the control in all ChIP assays. The DNA sequence at the exon 4-intron 4 junction (exon 4/intron 4) acts as a negative control region for all ChIP-qPCR assays. All experiments were performed independently in triplicate. * P

Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Mouse Assay, Sequencing, Negative Control

The enrichment of the enhancer protein complex at the promoter, intron 1 enhancer and intron 8 enhancer region during human erythroid progenitor CD34 + differentiation ex vivo . ( A–E ) Enrichment of GATA1, TAL1, LDB1, LMO2 and Pol II at the promoter, intron 1 enhancer and intron 8 enhancer region in differentiated erythroid cells at day 11, according to ChIP-qPCR. Normal rabbit IgG was employed as the control in all ChIP assays. The DNA sequence at the exon 4-intron 4 junction (exon 4/intron 4) acts as a negative control region for all ChIP-qPCR assays. All experiments were performed independently in triplicate. * P
Figure Legend Snippet: The enrichment of the enhancer protein complex at the promoter, intron 1 enhancer and intron 8 enhancer region during human erythroid progenitor CD34 + differentiation ex vivo . ( A–E ) Enrichment of GATA1, TAL1, LDB1, LMO2 and Pol II at the promoter, intron 1 enhancer and intron 8 enhancer region in differentiated erythroid cells at day 11, according to ChIP-qPCR. Normal rabbit IgG was employed as the control in all ChIP assays. The DNA sequence at the exon 4-intron 4 junction (exon 4/intron 4) acts as a negative control region for all ChIP-qPCR assays. All experiments were performed independently in triplicate. * P

Techniques Used: Ex Vivo, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Sequencing, Negative Control

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Incubation:

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Article Snippet: .. The gels were blotted onto a Hybond-P membrane (GE Health-care, Madrid, Spain) and incubated with the following antibodies (Abs): anti-NOS2, anti- IκB-α, anti-p65, anti-Sp1 (Santa Cruz Biotechnology, CA, USA), anti-P-ERK1/2, anti-P-Tyr705-STAT3 (Cell Signalling Technology Inc., MA, USA) and anti-α-actin (Sigma-Aldrich Co., St. Louis, USA). .. The blots were revealed by enhanced chemoluminiscence (ECL) in an Image Quant 300 cabinet (GE Healthcare Biosciences, PA, USA) following the manufacturer instructions.

Article Title: A synergistic interaction between transcription factors nuclear factor-?B and signal transducers and activators of transcription 3 promotes gastric cancer cell migration and invasion
Article Snippet: .. Equal amounts of proteins were loaded onto a 10% discontinuous SDS/polyacrylamide gel and electrophoretically transferred to PVDF membranes (Millipore Corporation, Billerica, MA, USA) blocked with 5% non-fat dry milk in phosphate-buffered saline-Tween-20 (0.1%, v/v) for 1 h. The membranes were then incubated at 4°C overnight with or without 2 h incubation at room temperature with one of the following primary antibodies: anti-RelA (1 : 1000, Santa Cruz Biotechnology), anti-phospho-Ser536-RelA (pRelA; 1:1000; Cell Signaling Technology), anti-STAT3 (1:500, Santa Cruz Biotechnology), anti-phospho-Tyr705-STAT3 (1:1000, Cell signaling Technology), anti-E-cadherin (1:1000, BD Biosciences, San Jose, CA, USA), anti-Snail (1:1000, Santa Cruz Biotechnology), anti-MMP9 (1:1000, Neomarkers), anti-β-actin (1:1000, Sigma, St Louis, MO, USA) and anti-TFIIB (1:1,000, BD Bioscience). .. Horseradish peroxidase-conjugated anti-rabbit IgG (1:2000, Zymed, San Francisco, CA, USA) or anti-mouse IgG (1:2500, Santa Cruz Biotechnology) was used as a secondary antibody.

Article Title: CBP Activity Mediates Effects of the Histone Deacetylase Inhibitor Butyrate on WNT Activity and Apoptosis in Colon Cancer Cells
Article Snippet: .. Two μg of either CBP (sc-369, Santa Cruz Biotechnology) or p300 (sc-584, Santa Cruz Biotechnology) antibody were added to the protein samples, and incubated overnight at 4o C with rotation. .. Fifty μL of a 50% protein A/G-agarose bead slurry (equilibrated in Co-IP buffer) were added, and after 1h-incubation at 4o C, the beads were washed three times (0.5 ml per wash) with Co-IP buffer and extracted with 30 μL of Laemmli buffer.

other:

Article Title: Human cytomegalovirus hijacks the autophagic machinery and LC3 homologs in order to optimize cytoplasmic envelopment of mature infectious particles
Article Snippet: Additional primary antibodies used in this study included anti-SQSTM1 (p62) (Abnova clone 2C11, H00008878-M01), anti-BECN1 (BD bioscience, 612112), anti-β-actin (Merck Millipore MAB1501 clone C4), anti-LC3 (MBL-PM036 and MBL-M152-3B), anti-LC3B (Sigma L7543, used for immunoblot analysis), anti-ULK1 (Santa Cruz, H-240 sc-33182),anti-GM130 (BD transduction, 610822), anti-GABARAPL2/Gate16 (R & D, 853746), anti-GABARAPL2/Gate16 (ProteinTech, 18724-1-AP), anti-GABARAPL1 (Cell-signaling, D5R9Y), anti-LAMP1 (Cell-signaling, D2D11), anti-EEA1 (Cell-signaling, C45B10), anti-ATG5 (sigma, A0731), anti-Gag/p41 (Abcam, ab63917) and DAPI (Invitrogen, D1306).

Article Title: Andrographolide inhibits the activation of NF-?B and MMP-9 activity in H3255 lung cancer cells
Article Snippet: Anti-p65, β-actin and anti-IκB antibodies were purchased from Santa Cruz Biotechnology, Inc., (Santa Cruz, CA, USA), while anti-phospho-IκB antibody was purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA).

Chromatin Immunoprecipitation:

Article Title: 1B/(-)IRE DMT1 Expression during Brain Ischemia Contributes to Cell Death Mediated by NF-?B/RelA Acetylation at Lys310
Article Snippet: .. To establish the effect of OGD on endogenous NF-κB/RelA interaction with 1B-DMT1 promoter and the associated histone acetylation, chromatin immunoprecipitation analysis was performed using anti-RelA and anti-acetylated histone H3 (H3K9/18ac) antibodies. .. Results illustrated in show that RelA binding to the NF-κB cis -acting element on 1B-DMT1 promoter increased in cortical neurons exposed to 3 hours OGD and 2 hours reoxygenation.

Purification:

Article Title: Dynamic recruitment of transcription factors and epigenetic changes on the ER stress response gene promoters
Article Snippet: .. Immunoprecipitations were performed with ProtG-Sepharose (KPL, USA) and 3–5 µg of the indicated antibodies: NF-YB [Purified rabbit polyclonal; Ref. ( )]; ATF6 (Active Motif 40962), C/EBPβ (Active Motif 39307), p53 (Active Motif 39041), Sp1 (Santa Cruz 59×), TBP [Purified mouse polyclonal; Ref. ( )], XBP-1 (Santa Cruz 7160), CHOP (Santa Cruz 575), p300 (Santa Cruz 585×), anti-Pol II (Santa Cruz 9001), Sp1 (Santa Cruz 59), acetyl-H3 (Upstate 06-599), acetyl-H4 (Upstate 06-759), H3-K4-me2 (Abcam 6000), H3-K4-me3 (Abcam 8580), H3-K79-me2 (Abcam 3594), H3-K9-me2 (Abcam 7312) and H3-K27-me3 (Abcam 6002). ..

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    Santa Cruz Biotechnology rabbit anti rna pol ii
    Knockdown of <t>EPHB3</t> rescues growth of TCF7L1-Null cells. ( A ) Immunohistochemistry for EPHB3 shows elevated expression in TCF7L1-Null xenograft tumors (bottom) compared to control tumors from the same mice (top), reflecting the significant mRNA upregulation observed by <t>RNA-sequencing</t> and qPCR. ( B ) EPHB3 knockdown largely rescued colony formation of TCF7L1-Null cells. Colony number and average colony size was significantly higher than that of TCF7L1-Null cells, and almost returned to the levels of control cells (*P
    Rabbit Anti Rna Pol Ii, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 90 stars, based on 2 article reviews
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    Santa Cruz Biotechnology rna pol ii
    Notch signaling represses Ccr9 transcription Chromatin immunoprecipitation was performed using antibodies against (A) <t>RNA</t> Polymerase II (PolII), (B) AcH3, (C) H3K4me3, and (D) H3K36me3 on extracts from 531026 cells isolated 48 hours after treatment with DMSO (grey) or GSI (black). The precipitated <t>DNA</t> was amplified by QPCR using primers located near the Ccr9 promoter (primers P1 and P2) or within the Ccr9 exons 1 (EX1), 2 (EX2) and 3 (EX3). Primers in a region −88.5 kb upstream of Ccr9 and at the Ebf1 or Deltex1 genes served as controls. Primers at the Deltex1 promoter were used for H3K4me3 and AcH3 and to Deltex1 exon 7 were used for H3K36me3. The data are expressed as enrichment normalized to input and are averaged from three independent experiments. * = p
    Rna Pol Ii, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Knockdown of EPHB3 rescues growth of TCF7L1-Null cells. ( A ) Immunohistochemistry for EPHB3 shows elevated expression in TCF7L1-Null xenograft tumors (bottom) compared to control tumors from the same mice (top), reflecting the significant mRNA upregulation observed by RNA-sequencing and qPCR. ( B ) EPHB3 knockdown largely rescued colony formation of TCF7L1-Null cells. Colony number and average colony size was significantly higher than that of TCF7L1-Null cells, and almost returned to the levels of control cells (*P

    Journal: Scientific Reports

    Article Title: TCF7L1 Modulates Colorectal Cancer Growth by Inhibiting Expression of the Tumor-Suppressor Gene EPHB3

    doi: 10.1038/srep28299

    Figure Lengend Snippet: Knockdown of EPHB3 rescues growth of TCF7L1-Null cells. ( A ) Immunohistochemistry for EPHB3 shows elevated expression in TCF7L1-Null xenograft tumors (bottom) compared to control tumors from the same mice (top), reflecting the significant mRNA upregulation observed by RNA-sequencing and qPCR. ( B ) EPHB3 knockdown largely rescued colony formation of TCF7L1-Null cells. Colony number and average colony size was significantly higher than that of TCF7L1-Null cells, and almost returned to the levels of control cells (*P

    Article Snippet: Gels were transferred onto nitrocellulose membranes and incubated overnight at 4C with one or more of the following antibodies: mouse anti-β-catenin (Sigma 15B8, 1:1,000), rabbit anti-TCF7L1 (Cell Signaling D15G11, 1:200), rabbit anti-LEF1 (Cell Signaling C12A5, 1:200), rabbit anti-TCF7 (Cell Signaling C63D9, 1:200), rabbit anti-TCF7L2 (Cell Signaling C48H11, 1:200), rabbit anti-EPHB3 (abcam EPR8280, 1:200), mouse anti-tubulin (Sigma T9026, 1:500), rabbit anti-RNA Pol II (Santa Cruz N-20, 1:200), mouse anti-GAPDH (Santa Cruz 6C5, 1:500), rabbit anti-Na + /K + -ATPase α (Santa Cruz H-300, 1:200).

    Techniques: Immunohistochemistry, Expressing, Mouse Assay, RNA Sequencing Assay, Real-time Polymerase Chain Reaction

    Notch signaling represses Ccr9 transcription Chromatin immunoprecipitation was performed using antibodies against (A) RNA Polymerase II (PolII), (B) AcH3, (C) H3K4me3, and (D) H3K36me3 on extracts from 531026 cells isolated 48 hours after treatment with DMSO (grey) or GSI (black). The precipitated DNA was amplified by QPCR using primers located near the Ccr9 promoter (primers P1 and P2) or within the Ccr9 exons 1 (EX1), 2 (EX2) and 3 (EX3). Primers in a region −88.5 kb upstream of Ccr9 and at the Ebf1 or Deltex1 genes served as controls. Primers at the Deltex1 promoter were used for H3K4me3 and AcH3 and to Deltex1 exon 7 were used for H3K36me3. The data are expressed as enrichment normalized to input and are averaged from three independent experiments. * = p

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Repression of Ccr9 transcription in mouse T lymphocyte progenitors by the Notch signaling pathway

    doi: 10.4049/jimmunol.1402443

    Figure Lengend Snippet: Notch signaling represses Ccr9 transcription Chromatin immunoprecipitation was performed using antibodies against (A) RNA Polymerase II (PolII), (B) AcH3, (C) H3K4me3, and (D) H3K36me3 on extracts from 531026 cells isolated 48 hours after treatment with DMSO (grey) or GSI (black). The precipitated DNA was amplified by QPCR using primers located near the Ccr9 promoter (primers P1 and P2) or within the Ccr9 exons 1 (EX1), 2 (EX2) and 3 (EX3). Primers in a region −88.5 kb upstream of Ccr9 and at the Ebf1 or Deltex1 genes served as controls. Primers at the Deltex1 promoter were used for H3K4me3 and AcH3 and to Deltex1 exon 7 were used for H3K36me3. The data are expressed as enrichment normalized to input and are averaged from three independent experiments. * = p

    Article Snippet: The protein-DNA complexes were detected using 5 μg of antibody against either RNA Pol II (N-20)(Santa Cruz, sc-899x), p300 (C-20)(Santa Cruz, sc-585x), HEB (A-20, Santa Cruz, sc-357) or E2A (N-649, Santa Cruz, sc-763x) in one ml of 0.8 M RIPA and incubated overnight at 4°C under rotating conditions.

    Techniques: Chromatin Immunoprecipitation, Isolation, Amplification, Real-time Polymerase Chain Reaction

    cAMP increases DGK activity, and SF1 synergizes with DGK to stimulate CYP17 transcriptional activity. (A and B) H295R cells were treated for 5 min to 2 h with 1 mM Bt 2 cAMP and nuclear extracts purified for analysis of DGK activity using the OG-DG mixed-micelle assay (see Materials and Methods). Radiolabeled PA produced was resolved by TLC (representative plate shown in panel A) and quantified by phosphorimager scanning and densitometry (panel B). (C) Expression of DGK isoforms in H295R cells was determined by real-time RT-PCR of control RNA (50 ng) and the PCRs resolved on a 2% agarose gel. (D) Cells were transfected with pGL3-CYP17-2x57, pRL-TK, pCR3.1-SF1, and DGK expression plasmids and then treated for 16 h with 1 mM Bt 2 cAMP. Data graphed are normalized to Renilla activity (pRL-TK) and expressed as n -fold increases of pGL3-CYP17-2x57 activity over pRL-TK activity of the untreated control group. Data shown represent the means ± standard errors of the means from at least three separate experiments, each performed in triplicate.

    Journal: Molecular and Cellular Biology

    Article Title: Cyclic AMP-Stimulated Interaction between Steroidogenic Factor 1 and Diacylglycerol Kinase ? Facilitates Induction of CYP17 ▿

    doi: 10.1128/MCB.00355-07

    Figure Lengend Snippet: cAMP increases DGK activity, and SF1 synergizes with DGK to stimulate CYP17 transcriptional activity. (A and B) H295R cells were treated for 5 min to 2 h with 1 mM Bt 2 cAMP and nuclear extracts purified for analysis of DGK activity using the OG-DG mixed-micelle assay (see Materials and Methods). Radiolabeled PA produced was resolved by TLC (representative plate shown in panel A) and quantified by phosphorimager scanning and densitometry (panel B). (C) Expression of DGK isoforms in H295R cells was determined by real-time RT-PCR of control RNA (50 ng) and the PCRs resolved on a 2% agarose gel. (D) Cells were transfected with pGL3-CYP17-2x57, pRL-TK, pCR3.1-SF1, and DGK expression plasmids and then treated for 16 h with 1 mM Bt 2 cAMP. Data graphed are normalized to Renilla activity (pRL-TK) and expressed as n -fold increases of pGL3-CYP17-2x57 activity over pRL-TK activity of the untreated control group. Data shown represent the means ± standard errors of the means from at least three separate experiments, each performed in triplicate.

    Article Snippet: The purified chromatin solutions were precleared with 1 μg rabbit or mouse immunoglobulin G and immunoprecipitated overnight at 4°C on a tube rotator using 5 μg of anti-acetyl histone H4, anti-GCN5, anti-RNA polymerase II (anti-Pol II), anti-SF1 or anti-SRC1, and protein A/G plus (Santa Cruz Biotechnology).

    Techniques: Activity Assay, Purification, Produced, Thin Layer Chromatography, Expressing, Quantitative RT-PCR, Agarose Gel Electrophoresis, Transfection

    High molecular mass complexes contain CBP and RNA pol II. Fractionation of T47D lysate on a Superose 6 column was analyzed by immunoblot with CBP and RNA Pol II-specific antibodies (CBP and RNA pol II). Recombinant baculovirus-expressed CBP also was fractionated (CBP BAC). Indicated are elution peaks of molecular mass markers: mammalian SWI/SNF complex (≈2 MDa) and thyroglobulin (670 kDa). The void volume (4 MDa for globular proteins) was determined at fraction 20 by silver staining after fractionation of T47D cell lysate (data not shown).

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Distinct steady-state nuclear receptor coregulator complexes exist invivo

    doi:

    Figure Lengend Snippet: High molecular mass complexes contain CBP and RNA pol II. Fractionation of T47D lysate on a Superose 6 column was analyzed by immunoblot with CBP and RNA Pol II-specific antibodies (CBP and RNA pol II). Recombinant baculovirus-expressed CBP also was fractionated (CBP BAC). Indicated are elution peaks of molecular mass markers: mammalian SWI/SNF complex (≈2 MDa) and thyroglobulin (670 kDa). The void volume (4 MDa for globular proteins) was determined at fraction 20 by silver staining after fractionation of T47D cell lysate (data not shown).

    Article Snippet: Commercially obtained antibodies used were anti-CBP (Upstate Biotechnologies, Lake Placid, NY), and anti-RNA polymerase II (RNA pol II) (Santa Cruz Biotechnology).

    Techniques: Fractionation, Recombinant, BAC Assay, Multiple Displacement Amplification, Silver Staining