Journal: Nature Communications

Article Title: RUNX3 regulates cell cycle-dependent chromatin dynamics by functioning as a pioneer factor of the restriction-point

doi: 10.1038/s41467-019-09810-w

Figure Lengend Snippet: The RUNX3–BRD2–nucleosome complex recruits SWI/SNF and TFIID. a Schematic diagram of BRD2 structure and interacting proteins. BD1 interacts with RUNX3 acetylated at Lys-94 and Lys-171; BD2 interacts with acetylated histones H4K4-ac, H4K12-ac, and H3K14-ac; and the C-terminal region interacts with the TFIID and SWI/SNF complexes. b , c HEK293 cells were serum-starved for 24 h, and then stimulated with 10% serum. Cells were harvested at the indicated time points, and the levels of the indicated proteins were measured by IP and IB. The time-dependent interactions were measured by IP and IB. d HEK293 cells were treated with control siRNA (si-con) or BRD2-specific siRNA (si-BRD2), serum-starved for 24 h, and then stimulated with 10% serum for the indicated durations. The time-dependent interactions between the proteins were measured by IP and IB. e HEK293 cells were transfected with Myc-RUNX3, Flag-BRD2-WT, Flag-BRD2-ΔCt (lacking C-terminal aa 633–802), Flag-BRD2-ΔBD1 (lacking BD1), or Flag-BRD2-ΔBD2 (lacking BD2). Cells were serum-starved for 24 h, and then stimulated with 10% serum. Cells were harvested after 2 h, and the interactions of the proteins were measured by IP and IB. f The RUNX3-binding site (GACCGCA) in the ARF enhancer region (ntd –1466) was deleted in HEK293 cells by the CRISPR/Cas9 method to obtain the HEK293-ARF-RX-D cell line. Deletion of the RUNX3-binding site was confirmed by nucleotide sequencing. Wild-type HEK293 cells (HEK293-ARF-WT) and HEK293-ARF-RX-D cells were serum-starved for 24 h. The cells were then treated with 10% serum, and the binding of the indicated proteins to the ARF promoter was measured by ChIP at the indicated time points. One-thirtieth of the lysates were PCR-amplified as input samples. g Schematic illustration of sequential molecular events at RUNX3 target loci during R-point regulation. RUNX3 binds to condensed chromatin marked by H3K27-me3 (inhibitory mark). p300 recruited to the loci acetylates RUNX3 and histones. Then, BRD2 binds both acetylated RUNX3 and acetylated histone through its two bromodomains. At 1 h after serum stimulation, SWI/SNF and TFIID are recruited to the loci through the C-terminal region of BRD2 to form Rpa-RX3-AC, and H3K27-me3 is replaced by H3K4-me3 (activating mark)

Article Snippet: Antibodies targeting HA (12CA5; dilution 1:1000; Cat# 11 666 606 001, Roche Applied Science, Mannheim, Germany), FLAG (M2; dilution 1:3000; Cat# F1804, Sigma, MO, USA), Myc (9E10; dilution 1:1000; Cat# sc-40, Santa Cruz Biotechnology), BRD2 (M01; dilution 1:1000; Cat# H00006046-M01, Abnova, Taipei City, Taiwan), pRB (dilution 1:1000; Cat# 554136, BD Biosciences, CA, USA), p-CDK4 (dilution 1:1000; Cat# PA5-64482, Invitrogen, CA, USA), MLL5 (dilution 1:1000; Cat# STJ27895, St. John’s Laboratory, London, UK) and MLL1 (dilution 1:1000; Cat# A300-374A, Bethyl Laboratories Inc., TX, USA) were used for IB and IP.

Techniques: Control, Transfection, Binding Assay, CRISPR, Sequencing, Amplification

Journal: Nature Communications

Article Title: RUNX3 regulates cell cycle-dependent chromatin dynamics by functioning as a pioneer factor of the restriction-point

doi: 10.1038/s41467-019-09810-w

Figure Lengend Snippet: RUNX3 sequentially recruits TrxG and PcG complexes. a Yeast two-hybrid screening using Gal4-BRD2 (aa 450–802) as bait identified RNF2 and MLL5 as BRD2-binding proteins (see STAR methods). DDO = SD-Leu/-Trp, DDO/X/A = SD-Leu/-Trp/X-α-gal/ABA, QDO/X/A = SD-Leu/-Trp/-His/-Ade/X-α-gal/ ABA medium. Selective colonies were identified by DNA sequencing. b HEK293 cells were serum-starved for 24 h, and then stimulated with 10% serum. Cells were harvested at the indicated time points, and the time-dependent interactions between RUNX3, BRD2, Cyclin D1, MLL1, MLL5, RNF2, BMI1, EZH2, EED, and HDAC4 were measured by IP and IB. c PLA showing RUNX3-MLL1 and RUNX3-MLL5 at the indicated time points after serum stimulation. Green fluorescence indicates association of the indicated proteins. F-actin was stained (red) to visualize the cytoplasmic compartment. d Microscopy images of transgenic fly eyes. Lozenge is a Drosophila homolog of the RUNX genes. Glass multimer reporter ( GMR ) -Gal4 promotes eye-specific expression of UAS -inserted genes. GMR -driven Lozenge overexpression ( GMR - Gal4/ + ;UAS-Lozenge (lz) → GMR > Lz) or GMR -driven Trithorax ( Trx ) overexpression ( GMR - Gal4/ + ; Trx G14137 → GMR > Trx) conferred weak rough phenotypes. However, GMR- driven overexpression of both Lz and Trx ( GMR > Lz + Trx ) resulted in a severe defective eye phenotype with loss of external ommatidial facets. e HEK293 cells were serum-starved for 24 h, and then stimulated with 10% serum. The binding of RUNX3, BRD2, MLL1, MLL5, CDK4, RNF2, Cyclin D1, HDAC4, EZH2, H2A-K119-Ub, H3K27-me3, and H3K4-me3 to the ARF promoter was measured by ChIP at the indicated time points. One-thirtieth of the lysates were PCR-amplified as input samples. f Schematic illustration of the R-point transition. At 1 h after serum stimulation, RUNX3 associates with various proteins, including p300, BRD2, H4K12-ac, SWI/SNF, TFIID, and MLL1/5, to form Rpa-RX3-AC. Between 2 and 4 h after serum stimulation, Rpa-RX3-AC interacts with PRC1–CyclinD1–HDAC4 to from a transient complex, Rpa-RX3-TR. Subsequently, Rpa-RX3-TR is destroyed (at 4 h) to form Rpa-RX3-RE (at 8 h)

Article Snippet: Antibodies targeting HA (12CA5; dilution 1:1000; Cat# 11 666 606 001, Roche Applied Science, Mannheim, Germany), FLAG (M2; dilution 1:3000; Cat# F1804, Sigma, MO, USA), Myc (9E10; dilution 1:1000; Cat# sc-40, Santa Cruz Biotechnology), BRD2 (M01; dilution 1:1000; Cat# H00006046-M01, Abnova, Taipei City, Taiwan), pRB (dilution 1:1000; Cat# 554136, BD Biosciences, CA, USA), p-CDK4 (dilution 1:1000; Cat# PA5-64482, Invitrogen, CA, USA), MLL5 (dilution 1:1000; Cat# STJ27895, St. John’s Laboratory, London, UK) and MLL1 (dilution 1:1000; Cat# A300-374A, Bethyl Laboratories Inc., TX, USA) were used for IB and IP.

Techniques: Two Hybrid Screening, Binding Assay, DNA Sequencing, Fluorescence, Staining, Microscopy, Transgenic Assay, Expressing, Over Expression, Amplification

Journal: Nature Communications

Article Title: RUNX3 regulates cell cycle-dependent chromatin dynamics by functioning as a pioneer factor of the restriction-point

doi: 10.1038/s41467-019-09810-w

Figure Lengend Snippet: Formation of the PRC1–CyclinD1–HDAC4 complex. a HEK293 cells were serum-starved for 24 h, and then stimulated with 10% serum. Cells were harvested at the indicated time points. Time-dependent formation of the RNF2–Cyclin D1, EZH2–Cyclin D1, HDAC4–Cyclin D1, and RNF2–HDAC4 complexes was measured by IP and IB. b HEK293 cells were transfected with HA-Cyclin D1, Myc-HDAC4, and Flag-RNF2, and the interactions between the proteins were measured by IP and IB. c , d HA-Cyclin D1, Myc-RNF2, and Myc-HDAC4 were translated in vitro and the interactions among the proteins were measured by IP and IB. e Regions of Cyclin D1 required for the interaction with RUNX3, RNF2, and HDAC4 are summarized. Cyclin D1 regions known to interact with pRB and CDK4/6 are also indicated. Cyc Box = Cyclin Box. f HEK293 cells were treated with control or HDAC4-specific siRNA (si-con or si-HDAC4), serum-starved for 24 h, and then stimulated with serum for the indicated durations. Time-dependent formation of the BRD2–RUNX3 complex was measured by IP and IB. g HEK293 cells were treated with control, RNF2-specific, or Cyclin D1-specific siRNA (si-con, si-RNF2, or si-CycD1), serum-starved for 24 h, and then stimulated with serum for the indicated durations. Time-dependent formation of the BRD2–RUNX3, Cyclin D1–RUNX3, HDAC4–RUNX3, and RNF2–BRD2 complexes was measured by IP and IB. h Schematic illustration of the process of Rpa-RX3-TR formation. Cyclin D1, which is induced 2 h after serum stimulation, interacts with PRC1 (containing RNF2) and matures into the PRC1–CyclinD1–HDAC4 complex. The PRC1–CyclinD1–HDAC4 complex then interacts with Rpa-RX3-AC to form Rpa-RX3-TR

Article Snippet: Antibodies targeting HA (12CA5; dilution 1:1000; Cat# 11 666 606 001, Roche Applied Science, Mannheim, Germany), FLAG (M2; dilution 1:3000; Cat# F1804, Sigma, MO, USA), Myc (9E10; dilution 1:1000; Cat# sc-40, Santa Cruz Biotechnology), BRD2 (M01; dilution 1:1000; Cat# H00006046-M01, Abnova, Taipei City, Taiwan), pRB (dilution 1:1000; Cat# 554136, BD Biosciences, CA, USA), p-CDK4 (dilution 1:1000; Cat# PA5-64482, Invitrogen, CA, USA), MLL5 (dilution 1:1000; Cat# STJ27895, St. John’s Laboratory, London, UK) and MLL1 (dilution 1:1000; Cat# A300-374A, Bethyl Laboratories Inc., TX, USA) were used for IB and IP.

Techniques: Transfection, In Vitro, Control

Journal: Nature Communications

Article Title: RUNX3 regulates cell cycle-dependent chromatin dynamics by functioning as a pioneer factor of the restriction-point

doi: 10.1038/s41467-019-09810-w

Figure Lengend Snippet: CDK4 plays key roles in the R-point transition. a HEK293 cells were serum-starved for 24 h, stimulated with 10% serum, and harvested at the indicated time points. Time-dependent formation of the BRD2–RUNX3, E2F1–RUNX3, CDK4–RUNX3, Cyclin D1–RUNX3, HDAC4–RUNX3, p16 INK4a –CDK4, p21–CDK4, Cyclin D1–CDK4, and HDAC4–CDK4 complexes was measured by IP and IB. Time-dependent phosphorylation of pRB (at Ser-795) and ERK1/2 was measured by IB. b PLA assay showing the RUNX3–CDK interaction 2 h after serum stimulation. c HEK293 cells were treated with control or CDK4-specific siRNA (si-con or si-CDK4), serum-starved for 24 h, and then stimulated with serum for the indicated durations. Time-dependent formation of the BRD2–RUNX3, CDK4–RUNX3, HDAC4–RUNX3, and Cyclin D1–RUNX3 complexes and phosphorylated RUNX3 were measured by IP and IB. Time-dependent expression of ARF was measured by IB. d HEK293 cells were treated with CDK4 inhibitor (PD0332991, 500 nM), serum-starved for 24 h, and then stimulated with serum for the indicated durations. Time-dependent formation of the BRD2–RUNX3 complex was measured by IP and IB. Time-dependent expression of ARF was measured by IB. e Cells were serum-starved for 24 h, and then stimulated with 10% serum. Cells were harvested at the indicated time points. Time-dependent RUNX3–CDK4 interaction and RUNX3 phosphorylation at Ser-356 were measured by IP and IB. f , g HEK293 cells were transfected with Myc-RUNX3, Myc-RUNX3-S356A, or Myc-RUNX3-S356E, serum-starved for 24 h, and then stimulated with 10% serum. Cells were harvested at the indicated time points. Time-dependent formation of the BRD2–RUNX3 complex, RUNX3 phosphorylation at Ser-356, and ARF expression were monitored by IP and IB. h Schematic illustration of the process of Rpa-RX3-AC → Rpa-RX3-TR transition. CDK4 of Rpa-RX3-AC and Cyclin D1 of PRC1–Cyclin D1–HDAC4 provide docking sites for the interaction of the two complexes, enabling formation of Rpa-RX3-TR

Article Snippet: Antibodies targeting HA (12CA5; dilution 1:1000; Cat# 11 666 606 001, Roche Applied Science, Mannheim, Germany), FLAG (M2; dilution 1:3000; Cat# F1804, Sigma, MO, USA), Myc (9E10; dilution 1:1000; Cat# sc-40, Santa Cruz Biotechnology), BRD2 (M01; dilution 1:1000; Cat# H00006046-M01, Abnova, Taipei City, Taiwan), pRB (dilution 1:1000; Cat# 554136, BD Biosciences, CA, USA), p-CDK4 (dilution 1:1000; Cat# PA5-64482, Invitrogen, CA, USA), MLL5 (dilution 1:1000; Cat# STJ27895, St. John’s Laboratory, London, UK) and MLL1 (dilution 1:1000; Cat# A300-374A, Bethyl Laboratories Inc., TX, USA) were used for IB and IP.

Techniques: Phospho-proteomics, Control, Expressing, Transfection

Journal: Nature Communications

Article Title: RUNX3 regulates cell cycle-dependent chromatin dynamics by functioning as a pioneer factor of the restriction-point

doi: 10.1038/s41467-019-09810-w

Figure Lengend Snippet: Multiple signals contribute to the R-point transition. a HEK293 cells were treated with MEK1 inhibitor (U0126, 1 μM). Time-dependent interactions of BRD2–RUNX3 and p300–RUNX3, as well as phosphorylation of ERK1/2, were monitored by IP and/or IB. b HEK293 cells were treated with control or CyclinD1-specific siRNA (si-con or si-CycD1). Time-dependent formation of the BRD2–RUNX3 and CDK4–RUNX3 complexes and phosphorylation of RUNX3 at Ser-356 and pRB at Ser-795 were measured by IP and IB. c Time-dependent formation of the JNK-CDK4 complexes and phosphorylations of RUNX3 at Ser-356 and CDK4 at Thr-172 were measured by IP and IB. d HEK293 cells were treated with JNK inhibitor (JNK-IN-8, 1 μM). Time-dependent formation of the BRD2–RUNX3 and RUNX3–CDK4 complexes and phosphorylation of RUNX3 at Ser-356 were measured by IP and IB. Time-dependent expression of ARF was measured by IB. e HEK293 cells were treated with control or JNK-specific siRNA (si-con or si-JNK). Time-dependent formation of the BRD2–RUNX3 and CDK4–RUNX3 complexes and phosphorylation of RUNX3 at Ser-356 were measured by IP and IB. Time-dependent expression of ARF was measured by IB. f HEK293 cells were transfected with Myc-RUNX3 and Flag-CDK4 WT or Flag-CDK4-T172A (CDK4 mutant defective in phosphorylation by JNK). Time-dependent formation of the BRD2–RUNX3 and CDK4–RUNX3 complexes and phosphorylation of RUNX3 at Ser-356 were measured by IP and IB. Time-dependent phosphorylation of pRB and expression of ARF were measured by IB. g HEK293 cells were treated with control or PIK3CA-specific siRNA (si-con or si- PIK3CA). Time-dependent formation of the BRD2–RUNX3 complex was measured by IP and IB. Time-dependent expression of ARF was measured by IB. h HEK293 cells were treated with control or mTORC1 inhibitor (Rapamycin, 100 nM). Time-dependent formation of the BRD2–RUNX3 complex was measured by IP and IB. Time-dependent expression of ARF was measured by IB. Ribosomal protein S6 kinase beta-1 (S6K1), which is phosphorylated by mTOR signaling, was used for control

Article Snippet: Antibodies targeting HA (12CA5; dilution 1:1000; Cat# 11 666 606 001, Roche Applied Science, Mannheim, Germany), FLAG (M2; dilution 1:3000; Cat# F1804, Sigma, MO, USA), Myc (9E10; dilution 1:1000; Cat# sc-40, Santa Cruz Biotechnology), BRD2 (M01; dilution 1:1000; Cat# H00006046-M01, Abnova, Taipei City, Taiwan), pRB (dilution 1:1000; Cat# 554136, BD Biosciences, CA, USA), p-CDK4 (dilution 1:1000; Cat# PA5-64482, Invitrogen, CA, USA), MLL5 (dilution 1:1000; Cat# STJ27895, St. John’s Laboratory, London, UK) and MLL1 (dilution 1:1000; Cat# A300-374A, Bethyl Laboratories Inc., TX, USA) were used for IB and IP.

Techniques: Phospho-proteomics, Control, Expressing, Transfection, Mutagenesis

Journal: Nature Communications

Article Title: RUNX3 regulates cell cycle-dependent chromatin dynamics by functioning as a pioneer factor of the restriction-point

doi: 10.1038/s41467-019-09810-w

Figure Lengend Snippet: R-point surveils aberrant oncogene activation. a HEK293 cells were transfected with empty vector (Vec) or Myc-K-RAS G12V . The time-dependent interactions among the components of Rpa-RX3-AC, Rpa-RX3-TR, and Rpa-RX3-RE were measured by IP and IB. Expression levels of ARF, p53, p21, and Myc-K-Ras G12V were measured by IB. b Binding of the components of Rpa-RX3-AC, Rpa-RX3-TR, and Rpa-RX3-RE to the p14 ARF promoter and histone marks (H4K12-ac, H3K27-me3, H3K4-me3, and H2A-K119-Ubi) at the locus were measured by ChIP at the indicated time points. One-thirtieth of the lysates were PCR-amplified as input samples. c Wild-type HEK293 cells (HEK293-ARF-WT) and HEK293-ARF-RX-D cells were transfected with empty vector (Vec) or Myc-K-Ras G12V . The binding of RUNX3, BRD2, H4K12-ac, H3K27-me3, and H3K4-me3 to the ARF promoter was measured by ChIP at the indicated time points. One-thirtieth of the lysates were PCR-amplified as input samples. d HEK293 cells were transfected with empty vector (Vec) or B-RAF V600E . Time-dependent formation of the RUNX3–p300 and BRD2–RUNX3 complexes was measured by IP and IB. Time-dependent expression of ARF and p53 was measured by IB. e HEK293 cells were transfected with empty vector (Vec) or Flag-MEK1-CA. Time-dependent interactions of CyclinD1–HDAC4 and CyclinD1–RNF2 were monitored by IP and IB. f Schematic illustration of differential regulation of the R-point in response to normal and oncogenic RAS. The RAS–RAF–MEK pathway inhibits formation of the PRC1/CyclinD1/HDAC4 complex, and thus inhibits the Rpa-RX3-AC → Rpa-RX3-TR transition. When the RAS–RAF–MEK pathway is activated by mitogenic stimulation, the activated pathway is downregulated after 4 h, allowing PRC1/CyclinD1/HDAC4 complex formation, which is followed by the Rpa-RX3-AC → Rpa-RX3-TR transition. If the RAF–MEK pathway is activated by oncogenic RAS, the constitutively activated signal inhibits formation of the PRC1/CyclinD1/HDAC4 complex for a long period of time, thereby inhibiting the Rpa-RX3-AC → Rpa-RX3-TR transition

Article Snippet: Antibodies targeting HA (12CA5; dilution 1:1000; Cat# 11 666 606 001, Roche Applied Science, Mannheim, Germany), FLAG (M2; dilution 1:3000; Cat# F1804, Sigma, MO, USA), Myc (9E10; dilution 1:1000; Cat# sc-40, Santa Cruz Biotechnology), BRD2 (M01; dilution 1:1000; Cat# H00006046-M01, Abnova, Taipei City, Taiwan), pRB (dilution 1:1000; Cat# 554136, BD Biosciences, CA, USA), p-CDK4 (dilution 1:1000; Cat# PA5-64482, Invitrogen, CA, USA), MLL5 (dilution 1:1000; Cat# STJ27895, St. John’s Laboratory, London, UK) and MLL1 (dilution 1:1000; Cat# A300-374A, Bethyl Laboratories Inc., TX, USA) were used for IB and IP.

Techniques: Activation Assay, Transfection, Plasmid Preparation, Expressing, Binding Assay, Amplification

Journal: Nature Communications

Article Title: RUNX3 regulates cell cycle-dependent chromatin dynamics by functioning as a pioneer factor of the restriction-point

doi: 10.1038/s41467-019-09810-w

Figure Lengend Snippet: The sequential molecular events for the R-point decision. a , b Upon mitogenic stimulation, RUNX3 binds to inactive chromatin marked by H3K27-me3. pRB–E2F1 and p300 associate with RUNX3. p300 acetylates RUNX3 and histones. BRD2 binds to acetylated RUNX3 through its first bromodomain (BD1). c One hour after mitogenic stimulation, the second bromodomain (BD2) of BRD2 binds to H4K12-ac: BRD2 binds both acetylated RUNX3 and acetylated histone through its bromodomains. Subsequently, SWI/SNF, MLL1/5, and TFIID bind to the C-terminal region of BRD2. At this point, inhibitory histone marks (H3K27-me3) are erased, and activatory marks (H3K4-me3) are enriched at the locus. Soon thereafter, TAF7 (inhibitory TAF) is released from the large complex, and expression of ARF, p53, and p21 is induced. The large complex, of which RUNX3 is the core, was named as Rpa-RX3-AC. d Two hours after mitogenic stimulation, CDK4 (associated with p21) binds to RUNX3 and becomes an additional component of Rpa-RX3-AC. At this point, the Cyclin D1–PRC1 complex forms separately from Rpa-RX3-AC. e When the RAS–MEK signal is downregulated, the Cyclin D1–PRC1 complex matures into the Cyclin D1–HDAC4–PRC1 complex, which in turn binds to Rpa-RX3-AC through the interaction between Cyclin D1 and CDK4 (a component of Rpa-RX3-AC), yielding Rpa-RX3-TR. Activation of CDK4 through the association with Cyclin D1 is critical for the inactivation of the chromatin loci and the dissociation of the entire complex. If the RAS signal is constitutively activated, the Cyclin D1–PRC1 complex fails to mature into the Cyclin D1–HDAC4–PRC1 complex, and consequently cannot form Rpa-RX3-TR. Therefore, if R-point commitment is normal, cells expressing constitutively active RAS cannot progress through the R-point into S-phase. f If the mitogenic signal is downregulated in a normal manner, Rpa-RX3-TR dissociates (4 h after stimulation) into two pieces, RUNX3–Cyclin D1–HDAC4 and BRD2–PRC1–SWI/SNF–TFIID, which remain associated with chromatin. g Soon thereafter, EZH2 associates with RUNX3–Cyclin D1–HDAC4 to form Rpa-RX3-RE, which remains on the chromatin. EZH2 contributes to the enrichment of an inactive chromatin mark (H3K27-me3) at the locus

Article Snippet: Antibodies targeting HA (12CA5; dilution 1:1000; Cat# 11 666 606 001, Roche Applied Science, Mannheim, Germany), FLAG (M2; dilution 1:3000; Cat# F1804, Sigma, MO, USA), Myc (9E10; dilution 1:1000; Cat# sc-40, Santa Cruz Biotechnology), BRD2 (M01; dilution 1:1000; Cat# H00006046-M01, Abnova, Taipei City, Taiwan), pRB (dilution 1:1000; Cat# 554136, BD Biosciences, CA, USA), p-CDK4 (dilution 1:1000; Cat# PA5-64482, Invitrogen, CA, USA), MLL5 (dilution 1:1000; Cat# STJ27895, St. John’s Laboratory, London, UK) and MLL1 (dilution 1:1000; Cat# A300-374A, Bethyl Laboratories Inc., TX, USA) were used for IB and IP.

Techniques: Expressing, Activation Assay

Journal: Nature Communications

Article Title: RUNX3 regulates cell cycle-dependent chromatin dynamics by functioning as a pioneer factor of the restriction-point

doi: 10.1038/s41467-019-09810-w

Figure Lengend Snippet: RUNX3 defends against endogenous oncogenic K-Ras. a H460-ERT2-RUNX3 and H460-ERT2-RUNX3-K94/171R cells were synchronized by serum deprivation and stimulated with 10% serum and 1 μM 4-OHT for the indicated durations (0, 4, and 8 h). Time-dependent subcellular localization of the expressed proteins was analyzed by double immunofluorescence staining (green = RUNX3; red = F-actin). b , c H460-ERT2-RUNX3 and H460-ERT2-RUNX3-K94/171R cells were serum-starved for 24 h, stimulated with 10% serum or 10% serum + 1 μM 4-OHT. Cells were harvested at the indicated time points, and the time-dependent interactions of RUNX3 with BRD2, p300, H4K12-ac, TFIID complex (TAF1, TAF7, and TBP), SWI/SNF complex (BRG-1 and BAF155), and MLL1/5 were measured by IP and IB. Expression of p14 ARF , p53, and p21 was measured by IB. The binding of the proteins and H4K12-ac, H3K27-me3, and H3K4-me3 to the ARF promoter was measured by ChIP at the indicated time points. One-thirtieth of the lysates were PCR-amplified as input samples. d H460-vec, H460-ERT2-RUNX3, and H460-ERT2-RUNX3-K94/171R cells were treated with indicated si-RNA, serum-starved for 24 h, and then stimulated with 10% serum or 10% serum + 1 μM 4-OHT for the indicated durations. Apoptotic cells were detected by flow cytometry after Annexin V–FITC/PI staining. The levels of p53 were measured by IB

Article Snippet: Antibodies targeting HA (12CA5; dilution 1:1000; Cat# 11 666 606 001, Roche Applied Science, Mannheim, Germany), FLAG (M2; dilution 1:3000; Cat# F1804, Sigma, MO, USA), Myc (9E10; dilution 1:1000; Cat# sc-40, Santa Cruz Biotechnology), BRD2 (M01; dilution 1:1000; Cat# H00006046-M01, Abnova, Taipei City, Taiwan), pRB (dilution 1:1000; Cat# 554136, BD Biosciences, CA, USA), p-CDK4 (dilution 1:1000; Cat# PA5-64482, Invitrogen, CA, USA), MLL5 (dilution 1:1000; Cat# STJ27895, St. John’s Laboratory, London, UK) and MLL1 (dilution 1:1000; Cat# A300-374A, Bethyl Laboratories Inc., TX, USA) were used for IB and IP.

Techniques: Double Immunofluorescence Staining, Expressing, Binding Assay, Amplification, Flow Cytometry, Staining

Journal: Bioorganic & medicinal chemistry

Article Title: Exploiting a water network to achieve enthalpy-driven, bromodomain-selective BET inhibitors

doi: 10.1016/j.bmc.2017.10.042

Figure Lengend Snippet: TR-FRET competitive binding data for exemplar BETi and THQ analogs 9–14.

Article Snippet: Time-resolved fluorescence energy transfer (TR-FRET) kits for BRD2-BD1 (catalog number 600500) and BRD2-BD2 (catalog number 600510) were was purchased from Cayman Chemical.

Techniques: Binding Assay

Journal: Bioorganic & medicinal chemistry

Article Title: Exploiting a water network to achieve enthalpy-driven, bromodomain-selective BET inhibitors

doi: 10.1016/j.bmc.2017.10.042

Figure Lengend Snippet: SPR results for 1 and THQ analogs 9–14.

Article Snippet: Time-resolved fluorescence energy transfer (TR-FRET) kits for BRD2-BD1 (catalog number 600500) and BRD2-BD2 (catalog number 600510) were was purchased from Cayman Chemical.

Techniques:

Journal: International Journal of Oncology

Article Title: Serum BRD2 autoantibody in hepatocellular carcinoma and its detection using mimotope peptide-conjugated BSA

doi: 10.3892/ijo.2022.5448

Figure Lengend Snippet: The target antigen of the XC246 autoantibody was identified as BRD2. (A) Preparative 10% SDS-PAGE was performed to isolate the XC246 antigen, and in-gel digestion was carried out for mass spectrometric-based protein identification. A preparative SDS-PAGE gel for western blotting was divided into two sections and blotted separately. The western blotting result is a combined image of two blots, with a dotted line representing the edges of two images. The protein band containing the XC246 antigen confirmed by western blotting was excised (indicated by the red arrow) and in-gel digested with trypsin. The proteins identified by mass spectrometric analysis are listed in . (B) Validation of the XC246 antigen as BRD2 by an RNA interference assay. HepG2 cells were transfected with siRNAs for candidate genes (EEF2, MYO1C and BRD2), and their cell lysates were examined by western blotting with the XC246 antibody. The knockdown of target genes was confirmed using reverse transcription polymerase chain reaction or western blotting. GAPDH was used as an internal control. (C) Immunoprecipitation analysis for the verification of the XC246 antigen as BRD2. The HepG2 cell lysate was immunoprecipitated with XC246 antibody-conjugated agarose beads and analyzed by western blotting with an anti-BRD2 or the XC246 antibody. Immunoprecipitates obtained using agarose beads without antibody conjugation were used as the control. Red arrows indicate the XC246 antigen or BRD2. (D) Immunofluorescence staining of the XC246 antigen in HepG2 cells. Fixed and permeabilized cells were treated with purified XC246 antibody or an anti-BRD2 antibody, followed by staining with FITC- or RDM-labeled anti-mouse IgG. To visualize the nuclei, cells were stained with DAPI. To verify the nuclear permeability of stained cells, an IgM-type mouse antibody (FBXO2 antibody) was also employed. (E) Western blot analysis of the intracellular distribution of the XC246 antigen or BRD2. Total cell lysates, subcellular fractions (cytosolic or nuclear fractions), and exosome lysates were prepared as described in the 'Materials and methods' and analyzed using western blotting. The blots were probed with the XC246 autoantibody, anti-BRD2 antibody, or anti-ATIC antibody. Each target antigen is indicated by colored arrows (red: XC246 and exosome XC246 antigen; blue: BRD2; green: ATIC). BRD2, bromodomain-containing protein 2; RDM, rhodamine; ATIC, AICAR transformylase/inosine monophosphate cyclohydrolase.

Article Snippet: The primary antibodies used in this study were as follows: BRD2 (Novus Biologicals, cat. no. NBP1-84310, NBP1-30475; 1:1,000 dilution), AICAR transformylase/inosine monophosphate cyclohydrolase (ATIC; Thermo Fisher Scientific, cat. no. MA1-086; 1:500 dilution), programmed cell death 6-interacting protein (ALIX; exosomal marker; Merck Millipore, cat. no. ABC1435; 1:500 dilution), calnexin (endoplasmic reticulum marker; Santa Cruz Biotechnology, cat. no. sc-46669; 1:1,000 dilution), GAPDH (Santa Cruz Biotechnology, cat. no. sc-47724; 1:5,000 dilution), and β-actin (Santa Cruz Biotechnology, cat. no. sc-8432; 1:5,000 dilution).

Techniques: SDS Page, Western Blot, Biomarker Discovery, Transfection, Knockdown, Reverse Transcription, Polymerase Chain Reaction, Control, Immunoprecipitation, Conjugation Assay, Immunofluorescence, Staining, Purification, Labeling, Permeability

Journal: International Journal of Oncology

Article Title: Serum BRD2 autoantibody in hepatocellular carcinoma and its detection using mimotope peptide-conjugated BSA

doi: 10.3892/ijo.2022.5448

Figure Lengend Snippet: Mass spectrometric analysis of XC246 antigen.

Article Snippet: The primary antibodies used in this study were as follows: BRD2 (Novus Biologicals, cat. no. NBP1-84310, NBP1-30475; 1:1,000 dilution), AICAR transformylase/inosine monophosphate cyclohydrolase (ATIC; Thermo Fisher Scientific, cat. no. MA1-086; 1:500 dilution), programmed cell death 6-interacting protein (ALIX; exosomal marker; Merck Millipore, cat. no. ABC1435; 1:500 dilution), calnexin (endoplasmic reticulum marker; Santa Cruz Biotechnology, cat. no. sc-46669; 1:1,000 dilution), GAPDH (Santa Cruz Biotechnology, cat. no. sc-47724; 1:5,000 dilution), and β-actin (Santa Cruz Biotechnology, cat. no. sc-8432; 1:5,000 dilution).

Techniques:

Journal: International Journal of Oncology

Article Title: Serum BRD2 autoantibody in hepatocellular carcinoma and its detection using mimotope peptide-conjugated BSA

doi: 10.3892/ijo.2022.5448

Figure Lengend Snippet: BRD2 autoantibody ELISA was developed using XC246p9 epitope-conjugated BSA for the detection of autoantibodies in human sera. (A) Biopanning of a phage-display random cyclic heptapeptide library for the isolation of epitope mimicries of the XC246 antigen. (B) Phage ELISA to confirm the binding specificity of the selected epitope mimicry phages to the XC246 autoantibody. M13 phages were coated with 10 11 pfu phages per well. Primary antibodies were used at the concentration of 0.1 µ g/well. Unrelated autoantibodies [K94 and XC90 ] were used as non-related controls. (C) Competitive FACS analysis of XC246 autoantibody binding to XC246 phages or HepG2 cells. Fixed and permeabilized cells (1×10 5 cells/reaction) were treated with the XC246 autoantibody (0.5 µ g). For antibody binding competition with the selected phages, cells were treated with the XC246 antibody pre-incubated with each phage (10 11 or 10 12 pfu/reaction), as indicated. (D) Preparation of the BSA-miniPEG2-XC246p9 antigen. A cyclic peptide with two miniPEG spacers, miniPEG2-XC246p9, was chemically synthetized and conjugated to bovine serum albumin (BSA) via amine-carboxyl acid coupling using the EDC reagent. The peptide-BSA conjugates (5 µ g/lane) were analyzed by SDS-PAGE and Coomassie blue staining. BSA-miniPEG2 without the epitope peptide was prepared as a control antigen. The synthetic peptide-conjugated to BSA was observed as a high-molecular-weight protein band. (E) ELISA with the BSA-miniPEG2-XC246p9 antigen. The antigen was coated at the indicated amount and detected with a gradually diluted XC246 autoantibody. (F) Competitive western blot analysis of XC246 autoantibody binding to the BSA-miniPEG2-XC246p9 antigen or tumor cell lysates. The cell lysates [HepG2 (H) or SNU638(S)] were loaded at a quantity of 15 µ g per lane, and BRD2 was detected with the XC246 autoantibody (1 µ g/10 ml). For the competitive inhibition of antibody binding to cell lysates, the XC246 antibody was pre-incubated with the BSA-miniPEG2-XC246p9 antigen (0.6 µ g/ml). BSA-miniPEG2-XC90p2 or BSA was used as the control competitor. The XC90p2 sequence is as follows: CPVRSGFPC. GAPDH was used as a loading control. BRD2, bromodomain-containing protein 2; BSA, bovine serum albumin.

Article Snippet: The primary antibodies used in this study were as follows: BRD2 (Novus Biologicals, cat. no. NBP1-84310, NBP1-30475; 1:1,000 dilution), AICAR transformylase/inosine monophosphate cyclohydrolase (ATIC; Thermo Fisher Scientific, cat. no. MA1-086; 1:500 dilution), programmed cell death 6-interacting protein (ALIX; exosomal marker; Merck Millipore, cat. no. ABC1435; 1:500 dilution), calnexin (endoplasmic reticulum marker; Santa Cruz Biotechnology, cat. no. sc-46669; 1:1,000 dilution), GAPDH (Santa Cruz Biotechnology, cat. no. sc-47724; 1:5,000 dilution), and β-actin (Santa Cruz Biotechnology, cat. no. sc-8432; 1:5,000 dilution).

Techniques: Enzyme-linked Immunosorbent Assay, Isolation, Binding Assay, Concentration Assay, Incubation, SDS Page, Staining, Control, High Molecular Weight, Western Blot, Inhibition, Sequencing

Journal: International Journal of Oncology

Article Title: Serum BRD2 autoantibody in hepatocellular carcinoma and its detection using mimotope peptide-conjugated BSA

doi: 10.3892/ijo.2022.5448

Figure Lengend Snippet: Human serum BRD2 autoantibody ELISA using BSA-miniPEG2-XC246p9 differentiated patients with HCC from non-HCC subjects. (A) AFP test and BRD2 autoantibody ELISA using BSA-miniPEG2-XC246p9 in sera of patients with HCC, as well as non-tumor subjects. The sample distribution was as follows: Control (n=91), HCC (n=118), cirrhosis (n=32) and benign liver cancer (n=3). Serum AFP levels were measured using a commercial quantification kit. The CV of AFP was 20 ng/ml, and the proportion of AFP-positive or -negative HCC (APHC or ANHC) is indicated within the box. The specific binding of the serum autoantibody to the XC246p9 epitope (anti-BRD2 response) was described as the difference in OD between the ELISA with BSA-miniPEG2-XC246p9 and that with BSA-miniPEG2. (B) The ROC curve analysis revealed the diagnostic sensitivity and specificity of each biomarker. All experiments were performed in duplicate and repeated at least three times. (C) Serum AFP and BRD2 autoantibody response related to tumor stage. The non-HCC group included control, cirrhosis, and benign liver cancer samples. (D) Serum AFP and BRD2 autoantibody response related to viral infection. The clinicopathological features of the participants are described in detail in . ns, not significant (P>0.05); BRD2, bromodomain-containing protein 2; BSA, bovine serum albumin; HCC, hepatocellular cancer; CV, cut-off value; APHC, AFP-positive HCC; ANHC, AFP-negative HCC; AFP, serum alpha-fetoprotein.

Article Snippet: The primary antibodies used in this study were as follows: BRD2 (Novus Biologicals, cat. no. NBP1-84310, NBP1-30475; 1:1,000 dilution), AICAR transformylase/inosine monophosphate cyclohydrolase (ATIC; Thermo Fisher Scientific, cat. no. MA1-086; 1:500 dilution), programmed cell death 6-interacting protein (ALIX; exosomal marker; Merck Millipore, cat. no. ABC1435; 1:500 dilution), calnexin (endoplasmic reticulum marker; Santa Cruz Biotechnology, cat. no. sc-46669; 1:1,000 dilution), GAPDH (Santa Cruz Biotechnology, cat. no. sc-47724; 1:5,000 dilution), and β-actin (Santa Cruz Biotechnology, cat. no. sc-8432; 1:5,000 dilution).

Techniques: Enzyme-linked Immunosorbent Assay, Control, Binding Assay, Diagnostic Assay, Biomarker Discovery, Infection

Journal: International Journal of Oncology

Article Title: Serum BRD2 autoantibody in hepatocellular carcinoma and its detection using mimotope peptide-conjugated BSA

doi: 10.3892/ijo.2022.5448

Figure Lengend Snippet: Combined analysis of serum autoantibody biomarkers with AFP enhanced the diagnostic accuracy of HCC. (A) Pearson's analysis of the correlations between the BRD2 autoantibody biomarker and the AFP or ATIC autoantibodies. The dotted lines represent the cutoff value of each biomarker diagnosis. The results of Pearson's analysis in individual cohorts are depicted in Fig S10. (B) Combined analysis of HCC biomarkers, AFP, BRD2 autoantibody, or ATIC autoantibody. The diagnostic values of each biomarker shown in panel A (AFP, anti-BRD2, and anti-ATIC) were simplified as either S-1 or S-0 according to whether their detection values surpassed or fail the cut-off value. Subsequently, the diagnostic values of each biomarker or their combination were analyzed. For the combined analysis of these markers, the unified diagnostic indexes of a serum sample were simply added and designated triple-negative samples as S-0, single-positive samples as S-1, double-positive samples as S-2, and triple-positive samples as S-3. The numbers on the plots represent the percentage of corresponding subjects. (C) Scattered plot analysis of HCC biomarker responses depending on AFP and autoantibody biomarker. The numbers in each quadrant represent the percentage of each case among patients with HCC (n=118). The numbers in the parentheses are the proportion of autoantibody biomarker-positive or -negative samples among ANHC or APHC cases. HCC, hepatocellular cancer; BRD2, bromodomain-containing protein 2; AFP, serum alpha-fetoprotein; ATIC, AICAR transformylase/inosine monophosphate cyclohydrolase; S-1, responsive; S-0, non-responsive; S-2, double positive; S-3, triple positive; ANHC, AFP-negative HCC; APHC, AFP-positive HCC.

Article Snippet: The primary antibodies used in this study were as follows: BRD2 (Novus Biologicals, cat. no. NBP1-84310, NBP1-30475; 1:1,000 dilution), AICAR transformylase/inosine monophosphate cyclohydrolase (ATIC; Thermo Fisher Scientific, cat. no. MA1-086; 1:500 dilution), programmed cell death 6-interacting protein (ALIX; exosomal marker; Merck Millipore, cat. no. ABC1435; 1:500 dilution), calnexin (endoplasmic reticulum marker; Santa Cruz Biotechnology, cat. no. sc-46669; 1:1,000 dilution), GAPDH (Santa Cruz Biotechnology, cat. no. sc-47724; 1:5,000 dilution), and β-actin (Santa Cruz Biotechnology, cat. no. sc-8432; 1:5,000 dilution).

Techniques: Diagnostic Assay, Biomarker Discovery

Journal: International Journal of Oncology

Article Title: Serum BRD2 autoantibody in hepatocellular carcinoma and its detection using mimotope peptide-conjugated BSA

doi: 10.3892/ijo.2022.5448

Figure Lengend Snippet: The clinicopathological features of the validation cohort a .

Article Snippet: The primary antibodies used in this study were as follows: BRD2 (Novus Biologicals, cat. no. NBP1-84310, NBP1-30475; 1:1,000 dilution), AICAR transformylase/inosine monophosphate cyclohydrolase (ATIC; Thermo Fisher Scientific, cat. no. MA1-086; 1:500 dilution), programmed cell death 6-interacting protein (ALIX; exosomal marker; Merck Millipore, cat. no. ABC1435; 1:500 dilution), calnexin (endoplasmic reticulum marker; Santa Cruz Biotechnology, cat. no. sc-46669; 1:1,000 dilution), GAPDH (Santa Cruz Biotechnology, cat. no. sc-47724; 1:5,000 dilution), and β-actin (Santa Cruz Biotechnology, cat. no. sc-8432; 1:5,000 dilution).

Techniques: Biomarker Discovery, Infection, Concentration Assay