human brd9  (Bethyl)

Journal: Journal of Lipid Research

Article Title: Chromatin remodeler BRD9 represses transcription of PPARα target genes, including CPT1A to suppress lipid metabolism

doi: 10.1016/j.jlr.2025.100874

Figure Lengend Snippet: Inhibition of BRD9 enhances the induction of PPARα-downstream gene CPT1A by WY-14643. A: Schematic image of mammalian cBAF, PBAF, and ncBAF . B: Glycerol gradient density sedimentation of the nuclear extract of HepG2 cells. Subunits of each SWI/SNF complex and PPARα protein were detected by Western blot analysis (C–G) HepG2, PHH, PMH, or HepaSH cells were pre-treated with 20 μM BI-9564. After 12 h, the cells were co-treated with 10 μM WY-14643 and 20 μM BI-9564 for 48 h. Human (C and E) and mouse (F) CPT1A mRNA levels were determined using real-time RT-PCR. (D) CPT1A, PPARα, and GAPDH protein levels in HepG2 cells were determined by Western blotting. Each column represents the mean ± SD (n = 4). Western blot experiments were conducted with three independent replicates. In panel (C), data were analyzed using one-way ANOVA followed by the Games-Howell test. In panel (F), data were analyzed using the Kruskal–Wallis test followed by Dunn’s test. Data in all other panels were analyzed by one-way ANOVA followed by Tukey’s test. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001, compared with NT. † P < 0.05, †† P < 0.01 and ††† P < 0.001, compared with BI-9564 (−). NT: non-treatment.

Article Snippet: Rabbit polyclonal antibody for human BRD9 (A303-781A) was purchased from Bethyl Laboratories.

Techniques: Inhibition, Sedimentation, Western Blot, Quantitative RT-PCR

Journal: Journal of Lipid Research

Article Title: Chromatin remodeler BRD9 represses transcription of PPARα target genes, including CPT1A to suppress lipid metabolism

doi: 10.1016/j.jlr.2025.100874

Figure Lengend Snippet: Effects of BRD7 and BRD9 knockdown on CPT1A expression in WY-14643-treated HepG2 cells. A–C: HepG2 cells were transfected with siRNA against BRD7 (siBRD7) or BRD9 (siBRD9). After 24 h, the cells were treated with 30 μM WY-14643 for 48 h. A: BRD7, BRD9, PPARα, and GAPDH protein levels were determined using Western blot analysis. B and C: CPT1A mRNA and protein levels were determined using real-time RT-PCR and Western blotting analysis, respectively. Each column represents the mean ± SD (n = 4). Western blot experiments were conducted with three independent replicates. Data in all panels were analyzed by one-way ANOVA followed by Tukey’s test. ∗∗ P < 0.01 and ∗∗∗ P < 0.001, compared with NT, † P < 0.05 and ††† P < 0.001, compared with siControl. NT: non-treatment.

Article Snippet: Rabbit polyclonal antibody for human BRD9 (A303-781A) was purchased from Bethyl Laboratories.

Techniques: Knockdown, Expressing, Transfection, Western Blot, Quantitative RT-PCR

Journal: Journal of Lipid Research

Article Title: Chromatin remodeler BRD9 represses transcription of PPARα target genes, including CPT1A to suppress lipid metabolism

doi: 10.1016/j.jlr.2025.100874

Figure Lengend Snippet: BRD9 directly interacts with PPARα. A: Co-immunoprecipitation assay to examine the interaction between PPARα and BRD9 was performed. HEK293T cells were transfected with FLAG-PPARα plasmid together with the BRD7-His or BRD9-His plasmids using Lipofectamine 3000. Cell lysates were subjected to co-immunoprecipitation with His-tag antibody. B: Direct interaction between PPARα and BRD9. HEK293T cells were individually transfected with FLAG-PPARα, BRD7-His, or BRD9-His plasmids. Affinity purification was conducted using cell lysates, and the purified FLAG-PPAR protein was incubated with purified BRD7/9-His proteins. C: Western blotting to analyze lysine acetylation of PPARα. Lysate of FLAG-PPARα plasmid-transfected HEK293T cells was subjected to Western blotting using anti-acetylated lysine and anti-FLAG antibodies. A smaller amount of protein (10 μg) was loaded compared to A (30 μg). Western blot experiments were conducted with three independent replicates.

Article Snippet: Rabbit polyclonal antibody for human BRD9 (A303-781A) was purchased from Bethyl Laboratories.

Techniques: Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation, Immunoprecipitation, Affinity Purification, Purification, Incubation, Western Blot

Journal: Journal of Lipid Research

Article Title: Chromatin remodeler BRD9 represses transcription of PPARα target genes, including CPT1A to suppress lipid metabolism

doi: 10.1016/j.jlr.2025.100874

Figure Lengend Snippet: Effects of WY-14643 and/or BI-9564 treatment on the binding of PPARα and BRD9 and on the chromatin accessibility around the CPT1A intronic peroxisomal proliferator response element (PPRE). A: Scheme of the CPT1A gene. B: Binding of PPARα to CPT1A intronic PPRE. HepG2 cells were pre-treated with 20 μM BI-9564. After 12 h, the cells were co-treated with 10 μM WY-14643 and 20 μM BI-9564 for 48 h. A ChIP assay using an anti-PPARα antibody was performed, and the purified DNA was analyzed by real-time PCR targeting CPT1A intronic PPRE. C: The interaction between PPARα and BRD9. HEK293T cells were transfected with FLAG-PPARα and BRD9-His plasmids using Lipofectamine 3000, and treated with 30 μM WY-14643 and/or 40 μM BI-9564 for 48 h. The cell lysates were co-immunoprecipitated with anti-His-tag antibody. FLAG-PPARα and BRD9-His were detected by Western blotting. D: Chromatin accessibility around CPT1A intronic PPRE. HepG2 cells were pre-treated with 20 μM BI-9564. After 12 h, the cells were co-treated with 10 μM WY-14643 and 20 μM BI-9564 for 48 h; a FAIRE assay was then performed on the cells. Purified DNA was analyzed by real-time PCR targeting the CPT1A intronic PPRE. Each column represents the mean ± SD (n = 3). Data in all panels were analyzed by one-way ANOVA followed by Tukey’s test. ∗∗∗ P < 0.001, compared with NT; † P < 0.05 and ††† P < 0.001, compared with BI-9564 (−). NT: non-treatment.

Article Snippet: Rabbit polyclonal antibody for human BRD9 (A303-781A) was purchased from Bethyl Laboratories.

Techniques: Binding Assay, Purification, Real-time Polymerase Chain Reaction, Transfection, Immunoprecipitation, Western Blot

Journal: Cell Reports Medicine

Article Title: Antitumor efficacy of a sequence-specific DNA-targeted γPNA-based c-Myc inhibitor

doi: 10.1016/j.xcrm.2023.101354

Figure Lengend Snippet:

Article Snippet: TaqManTM Gene Expression Assay (FAM) Inventoried human BRD9 (Hs01079464) , Thermofisher Scientific , Cat# 4331182.

Techniques: Plasmid Preparation, Recombinant, Binding Assay, Staining, Red Blood Cell Lysis, DC Protein Assay, Gene Expression, DNA Purification, Purification, Reverse Transcription, Luminex, RNA Sequencing, Amplification, Oligonucleotide Synthesis, Software, Fluorescence, Microscopy, Real-time Polymerase Chain Reaction, Electroporation, Multiplex Assay

Journal: bioRxiv

Article Title: The Energetics and Ion Coupling of Cholesterol Transport Through Patched1

doi: 10.1101/2023.02.14.528445

Figure Lengend Snippet: A) Coarse-grained (CG) and atomistic simulation setups of PTCH1 (light blue, PDB: 6RVD (CG)/6DMY (atomistic)) embedded in 3:1 POPC:cholesterol (white/purple) bilayers. Water is shown as transparent surface and Na + /Cl − ions are shown as blue/salmon spheres respectively. The inset shows the structure of cholesterol. B) Schematic diagram of the free energy changes associated with cholesterol movement between the PTCH1 sterol sensing domain (SSD) and the sterol binding domain (SBD) for the direct (ΔG 1 ) and indirect (ΔG 2 , ΔG 3 , ΔG 4 ) pathways. PTCH1-molA (yellow) and PTCH1-molB (light blue) are shown with either the ‘SHH-cholesterol’ (dark blue) or the ‘free cholesterol’ (purple) molecules positioned in the SBD and SSD (teal/ochre). The position of the extracellular (EC) and intracellular (IC) membrane leaflets are indicated in grey. C-E) CG potential of mean force (PMF) profiles for movement of ‘free cholesterol’ (purple) and ‘SHH-cholesterol’ (dark blue) between the SBD and the solvent (ΔG 4 ) ( C ) or the SSD and the bulk membrane (ΔG 2 ) ( E ) or for extraction of cholesterol from the membrane into the solvent (ΔG 3 ) ( D ). Bayesian bootstrapping (2000 rounds) was used to estimate profile errors (grey). Stars indicate the position of the ‘SHH-cholesterol’ (within PTCH1-molA) and ‘free cholesterol’ (within PTCH1-molB) densities in a revised cryo-EM structure (PDB: 6RVD ).

Article Snippet: Human Patched1 (hPtch1), comprising amino acids 75–1185 including a deletion of intracellular loop 3 (ICL3) (Δ630–717), was cloned into the pHR-CMV-TetO2 vector (Addgene plasmid #113893).

Techniques: Binding Assay, Membrane, Solvent, Extraction, Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: The Energetics and Ion Coupling of Cholesterol Transport Through Patched1

doi: 10.1101/2023.02.14.528445

Figure Lengend Snippet: A) Schematic diagram of the free energy changes associated with cholesterol movement between the SSD and SBD of PTCH1 for the direct pathway, coloured identically to in . Decoupling of cholesterol from the SBD/SSD sites are indicated by red circles, with the difference between them (ΔΔG 1 ) shown as a red arrow to indicate alchemical transformation. A black arrow indicates shows movement of cholesterol between the ECD base and the SBD, used in PMF calculations to derive ΔG 1 a. Grey arrows correspond to currently uncertain regions of the transport pathway. B) The free energy values of decoupling cholesterol from the SSD (PTCH1-molA) and SBD (PTCH1-molA and PTCH1-molB) as obtained from absolute binding free energy (ABFE) calculations. C) Snapshots from PMF-1a indicating movement of cholesterol between the ECD base and SBD of PTCH1 (residues used in the steered MD are labelled in red). CG representation of PTCH1 backbone beads are shown in transparent light blue/yellow, ‘free cholesterol’ is coloured purple, ‘SHH-cholesterol’ is coloured dark blue and lipid phosphate beads are shown in grey. D) PMF profile for cholesterol movement through the ECD of PTCH1-molA/B (ΔG 1 a) (see C ). Bootstrapping errors (2000 rounds) are shown in grey. The position of cholesterol within the SBD pocket in the cryo-EM model (PDB: 6RVD ) is starred. Arrows in D indicate energetic peaks 1–3 within the ECD core conserved between PTCH1-molA and PTCH1-molB (see ). E) HH signalling strength is determined by measuring endogenous Gli1 mRNA abundances (normalized to the control Gapdh) in response to SHH ligands (200 nM, 20 hours) in Ptch1 −/− cells stably expressing the indicated variants. Statistical significance is determined by a Student’s t-test with a Welch’s correction. Exact P values for comparisons: Ptch1 −/− untreated vs. SHH = 0.085, WT untreated vs. SHH < 0.0001, D513Y untreated vs. SHH = 0.2712, and P155R/N802E untreated vs. SHH = 0.0205. not significant (ns), * P > 0.05, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, and **** P ≤ 0.0001.

Article Snippet: Human Patched1 (hPtch1), comprising amino acids 75–1185 including a deletion of intracellular loop 3 (ICL3) (Δ630–717), was cloned into the pHR-CMV-TetO2 vector (Addgene plasmid #113893).

Techniques: Transformation Assay, Binding Assay, Cryo-EM Sample Prep, Control, Stable Transfection, Expressing

Journal: bioRxiv

Article Title: The Energetics and Ion Coupling of Cholesterol Transport Through Patched1

doi: 10.1101/2023.02.14.528445

Figure Lengend Snippet: Comparison of ΔG 1 values for ‘free cholesterol’ (purple) and ‘SHH-cholesterol’ (dark blue) movement between the PTCH1 SSD and SBD, derived from the indirect (PMF, ) and direct (ABFE, ) pathways. Step wise free energy changes are shown as connecting arrows and are labelled. ΔG 1 from the indirect pathway was calculated in quadrature since PMFs 2–4 were independent. In all cases cholesterol movement from the SSD to the SBD gives ΔG > 0 kJ mol −1 .

Article Snippet: Human Patched1 (hPtch1), comprising amino acids 75–1185 including a deletion of intracellular loop 3 (ICL3) (Δ630–717), was cloned into the pHR-CMV-TetO2 vector (Addgene plasmid #113893).

Techniques: Comparison, Derivative Assay

Journal: bioRxiv

Article Title: The Energetics and Ion Coupling of Cholesterol Transport Through Patched1

doi: 10.1101/2023.02.14.528445

Figure Lengend Snippet: A) Snapshot from atomistic simulations of PTCH1 (light blue, PDB: 6DMY ) indicating the location of cation binding sites within the TMD. Na + ions are shown as blue spheres and lipid phosphates are grey. Inset: cation-like density surrounded by anionic tired residues at Site 1 within the cryo-EM structure , as viewed from the extracellular face. A cylinder (length 4 nm, radius 1.3 nm) centred on the midpoint of V520 and I1092 Cα atoms is shown in grey and was used to identify water and ions within the PTCH1 TMD ( B) . B) The z coordinates of water oxygen atoms (light blue), Na + (blue) and Cl − (salmon) ions localised within the PTCH1 TMD (see A , grey cylinder) over the length of 3 × 100 ns simulations initiated with Na + bound at the density observed in A . Snapshots of Site 1 ( C ) and Site 2 ( D ) showing coordination of bound Na + ions by surrounding residues (stick representation) or waters (w 1–6 ). E) Free energy perturbation (FEP) calculations for alchemical transformation of Na + into K + within the solvent or bound to Site 1. F) Schematic representation of the free energy cycle used to calculate the difference in Na + binding to Site 1 compared to K + (ΔΔG).

Article Snippet: Human Patched1 (hPtch1), comprising amino acids 75–1185 including a deletion of intracellular loop 3 (ICL3) (Δ630–717), was cloned into the pHR-CMV-TetO2 vector (Addgene plasmid #113893).

Techniques: Binding Assay, Cryo-EM Sample Prep, Transformation Assay, Solvent

Journal: bioRxiv

Article Title: The Energetics and Ion Coupling of Cholesterol Transport Through Patched1

doi: 10.1101/2023.02.14.528445

Figure Lengend Snippet: Residue contacts with Na + bound at Site 1 across 3 × 100 ns simulations of PTCH1. Residues with < 1 % contact are excluded.

Article Snippet: Human Patched1 (hPtch1), comprising amino acids 75–1185 including a deletion of intracellular loop 3 (ICL3) (Δ630–717), was cloned into the pHR-CMV-TetO2 vector (Addgene plasmid #113893).

Techniques: Residue

Journal: bioRxiv

Article Title: The Energetics and Ion Coupling of Cholesterol Transport Through Patched1

doi: 10.1101/2023.02.14.528445

Figure Lengend Snippet: A) Time averaged water density (blue isosurface) across a 100 ns simulation of PTCH1 (PDB: 6DMY ) initiated with Na + bound at Site 1. PTCH1 TMD is shown in ribbon representation and anionic triad residues are shown as spheres. Yellow arrows indicate paths for water entry into the TMD. B) Residues comprising the hydrophobic cap (red box in A ) shown in stick and surface representation, as viewed from the extracellular (EC) face. V510 and I1092 forming part of the conserved GXXXDD and GXXX(E/D) motifs on TM4 and TM10 are boxed in blue. Residues mutated in disease phenotypes are boxed in black. C) Mean number of waters per frame within the EC half of the PTCH1 TMD across the final 10 ns of 3 × 50 ns simulations of WT PTCH1 or PTCH1 mutants (see ). D) HH signalling strength is determined by measuring endogenous Gli1 mRNA abundances (normalized to the control Gapdh) in response to SHH ligands (200 nM, 20 hours) in Ptch1 −/− cells stably expressing the indicated variants. Statistical significance is determined by a Student’s t-test with a Welch’s correction. Exact P values for comparisons: Ptch1 −/− untreated vs. SHH = 0.085, WT untreated vs. SHH < 0.0001, D513Y untreated vs. SHH = 0.2712, V510F untreated vs. SHH = 0.0002, L517C/P1125C untreated vs. SHH = 0.0255, and L570C/V1081C untreated vs. SHH = 0.0014. not significant (ns), * P > 0.05, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, and **** P ≤ 0.0001. E) Comparison of cation binding sites within PTCH1 and DISP1 (PDB: 7RPH ) TMDs. Anionic triad residues are shown as sticks. F) Time averaged water density profiles across 100 ns simulations of DISP1 in apo or ‘3x Na + ’ bound conformations. G) Mean number of waters per frame within the intracellular (IC) half of the DISP1 TMD across the final 10 ns of 3 × 100 ns simulations of DISP1 in apo and ‘3x Na + ’ bound states, or in ‘2x Na + ’, ‘1x Na + ’ and ‘0x Na + ’ bound states generated by sequential ion removal from the end of the previous Na + bound state or PTCH1 in apo, ‘3x Na + ’ and ‘3x K + ’ bound states (see ). C and G report the mean and standard deviation between repeats.

Article Snippet: Human Patched1 (hPtch1), comprising amino acids 75–1185 including a deletion of intracellular loop 3 (ICL3) (Δ630–717), was cloned into the pHR-CMV-TetO2 vector (Addgene plasmid #113893).

Techniques: Control, Stable Transfection, Expressing, Comparison, Binding Assay, Generated, Standard Deviation

Journal: bioRxiv

Article Title: The Energetics and Ion Coupling of Cholesterol Transport Through Patched1

doi: 10.1101/2023.02.14.528445

Figure Lengend Snippet: A) Proposed models for PTCH1 function (coloured as in ). Model-1: PTCH1 imports cholesterol extracted from SMO to a membrane sequestered pool or intracellular donor (energetically favourable). Model-2: Accessible cholesterol export by PTCH1 mediated by coupling to 1–3 Na + ions (blue, model-2a) or 2–3 K + ions (yellow, model-2b). Transition between ‘inward-open’ and ‘occluded’ states accompanied by breathing-like motions of intracellular helical segments (solid arrows) and alleviation of the hydrophobic cap (red). Model-3: PTCH1 re-partitions accessible cholesterol to an intramembrane sequestered pool (e.g. partnered with sphingomyelin) or intracellular acceptor. B) Free energy stored across the Na + (blue) and K + (yellow) membrane gradients as a function of membrane potential (ΔV) . C) Predicted number of Na + /K + coupling ions required per cholesterol exported by PTCH1 (defined as ‘cholesterol export free energy’/’free energy across cation potential’ from B ) vs membrane potential (ΔV). A cholesterol export free energy of +20 kJ mol −1 is indicated by a solid line, with export energies ranging between +10 to +40 kJ mol −1 indicated in transparent. Standard cellular ion concentrations ([Na + ] in : 12 mM, [Na + ] out : 145 mM, [K + ] in : 150 mM, [K + ] out : 4 mM) are assumed.

Article Snippet: Human Patched1 (hPtch1), comprising amino acids 75–1185 including a deletion of intracellular loop 3 (ICL3) (Δ630–717), was cloned into the pHR-CMV-TetO2 vector (Addgene plasmid #113893).

Techniques: Membrane