Journal: Pharmaceutics

Article Title: Controlled Delivery of BET-PROTACs: In Vitro Evaluation of MZ1-Loaded Polymeric Antibody Conjugated Nanoparticles in Breast Cancer

doi: 10.3390/pharmaceutics12100986

Figure Lengend Snippet: Average size, polydispersity index (PdI), and Z-potential of the different formulation obtained by dynamic light scattering (DLS).

Article Snippet: Zinc catalyst was prepared according to literature procedures [ ]. (+)-JQ1 based PROTAC (MZ1) were purchased (high-performance liquid chromatography (HPLC) ≥98% purity) by TOCRIS Bioscience (Bristol, UK), and trastuzumab was purchased as Herceptin by ROCHE (F. Hoffmann-La Roche Ltd.).

Techniques: Formulation

Journal: Pharmaceutics

Article Title: Controlled Delivery of BET-PROTACs: In Vitro Evaluation of MZ1-Loaded Polymeric Antibody Conjugated Nanoparticles in Breast Cancer

doi: 10.3390/pharmaceutics12100986

Figure Lengend Snippet: TEM images of ( A ) MZ1-NPs and ( B ) MZ1-ACNPs.

Article Snippet: Zinc catalyst was prepared according to literature procedures [ ]. (+)-JQ1 based PROTAC (MZ1) were purchased (high-performance liquid chromatography (HPLC) ≥98% purity) by TOCRIS Bioscience (Bristol, UK), and trastuzumab was purchased as Herceptin by ROCHE (F. Hoffmann-La Roche Ltd.).

Techniques:

Journal: Pharmaceutics

Article Title: Controlled Delivery of BET-PROTACs: In Vitro Evaluation of MZ1-Loaded Polymeric Antibody Conjugated Nanoparticles in Breast Cancer

doi: 10.3390/pharmaceutics12100986

Figure Lengend Snippet: ( A ) Physical stability of ACNPs. ( B ) In vitro release profiles of MZ1-NPs and MZ1-ACNPs at pH 7.4. Data are expressed as mean ± s.e.m. from at least three independent experiments.

Article Snippet: Zinc catalyst was prepared according to literature procedures [ ]. (+)-JQ1 based PROTAC (MZ1) were purchased (high-performance liquid chromatography (HPLC) ≥98% purity) by TOCRIS Bioscience (Bristol, UK), and trastuzumab was purchased as Herceptin by ROCHE (F. Hoffmann-La Roche Ltd.).

Techniques: In Vitro

Journal: Pharmaceutics

Article Title: Controlled Delivery of BET-PROTACs: In Vitro Evaluation of MZ1-Loaded Polymeric Antibody Conjugated Nanoparticles in Breast Cancer

doi: 10.3390/pharmaceutics12100986

Figure Lengend Snippet: Trastuzumab vectorization increases the antitumor efficacy of MZ1-carrying nanoparticles. Cell viability (in %, referred to the DMSO vehicle) by MTT assay under treatment with control (Ctl) free MZ1, MZ1-NPs or MZ1-ACNPs in SKBR3 ( A ) and BT474 ( B ) HER2+ cell lines. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Zinc catalyst was prepared according to literature procedures [ ]. (+)-JQ1 based PROTAC (MZ1) were purchased (high-performance liquid chromatography (HPLC) ≥98% purity) by TOCRIS Bioscience (Bristol, UK), and trastuzumab was purchased as Herceptin by ROCHE (F. Hoffmann-La Roche Ltd.).

Techniques: MTT Assay, Control

Journal: Pharmaceutics

Article Title: Controlled Delivery of BET-PROTACs: In Vitro Evaluation of MZ1-Loaded Polymeric Antibody Conjugated Nanoparticles in Breast Cancer

doi: 10.3390/pharmaceutics12100986

Figure Lengend Snippet: MZ1-encapsulated nanoparticles do not affect cell cycle distribution in HER2+ cell lines and increase cell death by apoptosis induction in HER2+ cell lines. Distribution of cells (in % of the total) under treatment with vehicle, free MZ1, MZ1-NPs, or MZ1-ACNPs in SKBR3 ( A , C ) and BT474 ( B , D ) HER2+ cell lines, evaluated by flow cytometry. ** p < 0.01; *** p < 0.001.

Article Snippet: Zinc catalyst was prepared according to literature procedures [ ]. (+)-JQ1 based PROTAC (MZ1) were purchased (high-performance liquid chromatography (HPLC) ≥98% purity) by TOCRIS Bioscience (Bristol, UK), and trastuzumab was purchased as Herceptin by ROCHE (F. Hoffmann-La Roche Ltd.).

Techniques: Flow Cytometry

Journal: Pharmaceutics

Article Title: Controlled Delivery of BET-PROTACs: In Vitro Evaluation of MZ1-Loaded Polymeric Antibody Conjugated Nanoparticles in Breast Cancer

doi: 10.3390/pharmaceutics12100986

Figure Lengend Snippet: MZ1-ACNPs rendered a strong cytotoxic effect in trastuzumab, MZ1-naturally resistant cell line HCC1954. Cell viability (in %, referred to the DMSO vehicle) by MTT assay under treatment with free MZ1, MZ1-NPs, or MZ1-ACNPs. *** p < 0.001.

Article Snippet: Zinc catalyst was prepared according to literature procedures [ ]. (+)-JQ1 based PROTAC (MZ1) were purchased (high-performance liquid chromatography (HPLC) ≥98% purity) by TOCRIS Bioscience (Bristol, UK), and trastuzumab was purchased as Herceptin by ROCHE (F. Hoffmann-La Roche Ltd.).

Techniques: MTT Assay

Journal: Molecular cancer research : MCR

Article Title: BET Proteins Exhibit Transcriptional and Functional Opposition in the Epithelial-to-mesenchymal Transition

doi: 10.1158/1541-7786.MCR-17-0568

Figure Lengend Snippet: Individual BET proteins control independent EMT transcriptomes. A, Heatmap presenting Z scores of a PCR array of 84 EMT genes expressed in MDA-MB-231 cells upon BET protein depletion by siRNA (50 nM for three days) (n=3). Independent transcriptional signatures relevant to EMT regulation were obtained for each BET protein. Pan-BET inhibition using small molecule JQ1 (400 nM for three days) obscured these individual profiles. A color code is used to illustrate Z score variations. Normalization is set to scramble.

Article Snippet: JQ1 was purchased from Tocris Bioscience.

Techniques: Control, Inhibition

Journal: bioRxiv

Article Title: Microfluidic Agarose µ -Droplets for DNA-Encoded Chemical Library Screening

doi: 10.64898/2026.02.15.706034

Figure Lengend Snippet: (A) Chemical structure of the JQ1-oligo-Cy5 conjugate used as a fluorescent probe (BP). JQ1, a selective BRD4 ligand, is covalently linked to a DNA oligonucleotide and a Cy5 fluorophore, allowing fluorescence-based detection of BRD4 binding. (B) Schematic illustrations (top) and corresponding fluorescence images (bottom) of agarose μ-droplets containing magnetic beads only, BRD4-bound magnetic beads, agarose alone, intact HeLa cells, or permeabilized HeLa cells after incubation with JQ1-oligo-Cy5. Strong Cy5 fluorescence was observed in droplets containing BRD4-bound magnetic beads and permeabilized HeLa cells, whereas no detectable Cy5 fluorescence was observed in magnetic beads only, intact HeLa cells, and agarose-only droplets. Scale bars, 50μm. (C) Schematic illustration of two-color Exchange-PAINT imaging using orthogonal DNA docking-imager strand pairs. An R6 docking strand conjugated to an anti-GFP nanobody was used to label GFP-BRD4, while an R2 docking strand was incorporated into the JQ1-based probe (JQ1-BP). Sequential super-resolution imaging was performed using R6* and R2* imager strands to independently localize BRD4 and bound JQ1, respectively. (D) Two-color Exchange-PAINT super-resolution images of GFP-BRD4-expressing Cos7 cells showing GFP-BRD4 (green) and the JQ1-based probe (JQ1-BP, red). Left, whole-nucleus view; middle, magnified view of the boxed region highlighting nanoscale clustering of BRD4 and JQ1. Right, the same region after Q-PAINT-based cluster filtering (K > 10), revealing higher-order nanoclusters containing both BRD4 and JQ1. Enrichment of JQ1-BP localizations at GFP-BRD4 clusters indicates that JQ1 preferentially localizes to BRD4-enriched nuclear regions.

Article Snippet: Ni–NTA agarose resin and disposable gravity columns were purchased from Qiagen (Venlo, Nether-lands). (+)-JQ1 maleimide (catalog no. 7576) was obtained from Tocris Bioscience (Bristol, UK), AccuPower® ProFi Taq PCR PreMix and Accu-Power® 2X GreenStarTM qPCR MasterMix were obtained from Bioneer (Daejeon, Korea).

Techniques: Fluorescence, Binding Assay, Magnetic Beads, Incubation, Imaging, Expressing

Journal: bioRxiv

Article Title: Microfluidic Agarose µ -Droplets for DNA-Encoded Chemical Library Screening

doi: 10.64898/2026.02.15.706034

Figure Lengend Snippet: (A) Composition of the four-compound library used for proof-of-concept DEL screening, including JQ1 as a positive control, GL-CBS and methotrexate (MTX) as off-target binders, and benzoic acid as a negative control. (B) Enrichment ratios (Target / Control) derived from nanopore sequencing data following small-scale DEL screening in agarose μ-droplets containing BRD4-bound magnetic beads (target) or magnetic beads only (control). JQ1 exhibited strong enrichment above the threshold (Target / Control = 1), whereas other compounds showed minimal enrichment. (C) Quantitative analysis of small-scale DEL screening performed in cell-containing agarose μ-droplets, in which DEL enrichment was quantified by qPCR following screening. Agarose-only μ-droplets were used as a control condition, while μ-droplets containing permeabilized HeLa cells or BRD4-overexpressing HeLa cells were treated as target conditions. JQ1 showed the highest enrichment among tested compounds and displayed a pronounced preference for BRD4-overexpressing HeLa cells compared to permeabilized cells. Representative bright field, emGFP, and Cy5 fluorescence images are shown on the right. Scale bars, 50μm. emGFP fluorescence was observed specifically in BRD4-overexpressing HeLa cells. As both HeLa cell conditions were permeabilized, Cy5 fluorescence originating from BP was detected in droplets containing either permeabilized or BRD4-overexpressing HeLa cells.

Article Snippet: Ni–NTA agarose resin and disposable gravity columns were purchased from Qiagen (Venlo, Nether-lands). (+)-JQ1 maleimide (catalog no. 7576) was obtained from Tocris Bioscience (Bristol, UK), AccuPower® ProFi Taq PCR PreMix and Accu-Power® 2X GreenStarTM qPCR MasterMix were obtained from Bioneer (Daejeon, Korea).

Techniques: Drug discovery, Positive Control, Negative Control, Control, Derivative Assay, Nanopore Sequencing, Magnetic Beads, Fluorescence

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Bromodomain-Containing Protein 4 (BRD4) Inhibition Sensitizes Palomid 529-Induced Anti-Renal Cell Carcinoma Cell Activity in Vitro and in Vivo.

doi: 10.1159/000494185

Figure Lengend Snippet: Fig. 7. JQ1 facilitates Palomid 529-induced anti-tumor activity in vivo. The 786-O xenograft tumor-bearing SCID mice were treated with vehicle control (“Vehicle”), Palomid 529 (“P529”, 100 mg/kg/2 d, i.p., for 18 days) and/or JQ1 (50 mg/kg/2 d, i.p., for 18 days), the tumor volume (A) and mice body weight (D) were recorded every 6 days for a total of 42 days; Estimated daily tumor growth was calculated as described (B); At Day-42, tumors of each group were isolated and weighted (C); At Day-6, three hours after the drug administration, one tumor of each group was isolated, listed signaling proteins were tested by Western blotting assay of tumor lysates (E). For each group, n=10. * p<0.05 vs. “Vehicle” group. # p<0.05 vs. “P529” only group.

Article Snippet: Materials and Methods Reagents and antibodies Palomid 529, JQ1, MS436 and CPI203 were purchased from Selleck (Shanghai, China).

Techniques: Activity Assay, In Vivo, Control, Isolation, Western Blot

Journal: Clinical & Translational Immunology

Article Title: Integration of single‐cell and bulk transcriptomics reveals the association of manganese metabolism‐related genes with prognosis and immune infiltration in lung adenocarcinoma

doi: 10.1002/cti2.70085

Figure Lengend Snippet: Validation of potential drugs for PTMA. (a) Prediction of potential drugs for high‐ and low‐PTMA expression groups. Blue indicates samples in the low‐PTMA group, while red indicates samples in the high‐PTMA group. The y‐axis represents the drug sensitivity score, with lower scores indicating higher sensitivity to the corresponding drug. (b) CCK8 assay was used to detect the effects of JQ1 and PTMA expression on tumor cell proliferation. (c) Transwell assay was used to detect the effects of JQ1 and PTMA expression on tumor cell invasion. (d) Clonogenic assay was used to detect cell clonal levels. (e) Scratch wound healing assay was used to detect cell migration ability. (f) Flow cytometry was used to detect apoptosis levels. (g) Western blot assay was used to detect the expression of apoptosis‐related proteins. ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: JQ1 was purchased from MCE Biotechnology Co., Ltd. (catalogue number HY‐13030) and was used to treat lung cancer cells at a concentration of 1 μM.

Techniques: Biomarker Discovery, Expressing, CCK-8 Assay, Transwell Assay, Clonogenic Assay, Wound Healing Assay, Migration, Flow Cytometry, Western Blot

Journal: Cell communication and signaling : CCS

Article Title: Investigation of the impact of bromodomain inhibition on cytoskeleton stability and contraction.

doi: 10.1186/s12964-024-01553-6

Figure Lengend Snippet: Fig. 2 Pattern and enrichment analyses support JQ1 attenuation of genes associated with cytoskeletal changes. Four publicly available datasets in which mechanically active cells were stimulated with a physiologically relevant pro-fibrotic condition in the absence or presence of JQ1 were identified and reanalyzed. Differential gene expression analysis was performed using DESeq2 after generating count matrices from the fastq files using Biogrids software tools including: SRAtoolkit, STAR, Subread. A, C, E Pattern analysis of DEGs from each of the four datasets identified 2 groups of genes that were perturbed by PDGF (GSE11714), Growth medium (GSE138323), or TGFB (GSE127229) and attenuated by JQ1. B, D, F JQ1 sensitive genes were subjected to GO cellular compartment terms and clustered based on common genes between terms. Highlighted in yellow are terms related to chromatin remodeling. Highlighted in green are terms related to cytoskeleton regulation

Article Snippet: Validation of in silico predicted JQ1 sensitive genes via immunoblots Targets assessed via immunoblot included PDGFRβ, PDGFRα, ITGB1, ITGA5, αACTN, SRC, and CFL (Cell Signaling Technology).

Techniques: Gene Expression, Software

Journal: Cell communication and signaling : CCS

Article Title: Investigation of the impact of bromodomain inhibition on cytoskeleton stability and contraction.

doi: 10.1186/s12964-024-01553-6

Figure Lengend Snippet: Fig. 3 JQ1 and BRD4 knock-down converge on Myc and AP1 to attenuate cytoskeleton related genes. An additional dataset comprising IMR90, Human airway fibroblasts, under conditions of quiescence or growth and stimulated with DMSO, JQ1, control shRNA, or shBRD4. Pattern analysis was performed to identify JQ1 sensitive and BRD4 sensitive DEGs defined as those that were perturbed by growth conditions and attenuated to near baseline, quiescent levels with JQ1 or shBRD4. A A Venn diagram illustrating the extent of shared DEGs that were sensitive to both JQ1 and shBRD4. B Genes from each group in the Venn diagram; attenuated only by JQ1, attenuated only by shBRD4, and those attenuated by both were subjected to TF enrichment analysis with ChEA3 using the ENCODE, ChIP-seq database of TF-gene interactions was used to identify TFs implicated in regulated each group of genes. TFs were rank-ordered by the –log10 of the Fisher’s Exact Test (FET) value. C Normalized counts of relevant TFs implicated in the TF enrichment analysis, TFs implicated in previously published findings as well as bromodomains were plotted. Each plot contains the average expression of DMSO-stimulated quiescent cells (Ctrl_Qsnt), DMSO or shRNA control stimulated proliferative cells (Ctrl_Pro), and JQ1 or shBRD4 stimulated proliferative cells (Expt_Pro) in pink and blue respectively. JQ1 attenuated DEGs and shBRD4 attenuated DEGs were separated between up regulated and down regulated genes (based on their expression in JQ1 and shBRD4 proliferative cells compared to the proliferative control cells). The down regulated, JQ1 and shBRD4 genes were subjected to active subnetwork searches to BIOGRID protein– protein interaction network and subsequent enrichment analysis using GO terms for cellular compartment with the pathfindR package. D The shared and uniquely enriched terms between the shBRD4 (BRD4) attenuated DEGs and the JQ1 attenuated DEGs were plotted in a dotplot. Each dot reflects an enriched term, the x-axis reflects the fold enrichment of each term, the color of each dot corresponds to the –log10 of the adjusted p-value which is adjusted based on Bonferroni method and the size of each dot corresponds to the number of genes from the DEG lists associated with each term. Cytoskeleton associated terms sensitive to both JQ1 and BRD4 knock-down are highlighted with green rectangles

Article Snippet: Validation of in silico predicted JQ1 sensitive genes via immunoblots Targets assessed via immunoblot included PDGFRβ, PDGFRα, ITGB1, ITGA5, αACTN, SRC, and CFL (Cell Signaling Technology).

Techniques: Knockdown, Control, shRNA, ChIP-sequencing, Expressing

Journal: Cell communication and signaling : CCS

Article Title: Investigation of the impact of bromodomain inhibition on cytoskeleton stability and contraction.

doi: 10.1186/s12964-024-01553-6

Figure Lengend Snippet: Fig. 4 Validation of JQ1 sensitive cytoskeletal effectors via qPCR and immunoblots. A subset of cytoskeleton associated targets predicted to be sensitive to JQ1 were validated using qPCR and immunoblotting techniques. Primers for JQ1 sensitive cytoskeleton associated targets were generated using NCBI primer design tool. A time course of vehicle (V), PDGF (P), or TGFB (T) stimulated RBMC with concurrent stimulation of DMSO or JQ1 was performed. A The average Log2FC of three replicates was plotted in a heat map. B The same targets were assessed by immunoblot at 16 h and C quantified. Data are representative of 3 independent trials

Article Snippet: Validation of in silico predicted JQ1 sensitive genes via immunoblots Targets assessed via immunoblot included PDGFRβ, PDGFRα, ITGB1, ITGA5, αACTN, SRC, and CFL (Cell Signaling Technology).

Techniques: Biomarker Discovery, Western Blot, Generated

Journal: Cell communication and signaling : CCS

Article Title: Investigation of the impact of bromodomain inhibition on cytoskeleton stability and contraction.

doi: 10.1186/s12964-024-01553-6

Figure Lengend Snippet: Fig. 5 In vitro functional validation of JQ1 attenuation of PDGF and TGFB stimulated contraction in RBMC. A Representative images of RBMC stimulated with PBS (Veh), PDGF, TGFB with DMSO or JQ1 for 16 h and stained with Vimentin, Phalloidin and DAPI. B Phalloidin intensity and cell shape was quantified in ImageJ. C RBMC were plated on 1.2 mg/mL collagen gels in a 12 well plate in 1 mL of serum free media and incubated for 16 h with PBS (Veh), PDGF, TGFB with DMSO or JQ1. Select wells were stimulated for 30 min with a ROCK inhibitor as a negative control for contraction. Collagen gels were separated from the walls of the wells and Imaged after 1.5 h to capture spontaneous (unstimulated contraction, followed by an additional 1.5 h in 5% FBS to facilitate contraction. D Contraction was quantified using ImageJ and measured as a percent of the changed area of the gel from the baseline. Data are representative of 3 independent trials. 1NC_FBS refers to negative control for FBS in which cells never receive FBS from platting to harvesting. 2NC_iROCK refers to the negative control for contraction in which cells are stimulated with a ROCK inhibitor for 30 min prior to observing contraction. *, p < 0.05, ** p < 0.01, ***, p < 0.001, ****, p < 0.0001 compared to control. #, p < 0.05, ## p < 0.01, ###, p < 0.001, ###, p < 0.0001 compared to PDGF. $, p < 0.05, $$ p < 0.01, $$$, p < 0.001, $$$$, p < 0.0001 compared to TGFB. Statistical significance was calculated with student t-test

Article Snippet: Validation of in silico predicted JQ1 sensitive genes via immunoblots Targets assessed via immunoblot included PDGFRβ, PDGFRα, ITGB1, ITGA5, αACTN, SRC, and CFL (Cell Signaling Technology).

Techniques: In Vitro, Functional Assay, Biomarker Discovery, Staining, Incubation, Negative Control, Control

Journal: Cell communication and signaling : CCS

Article Title: Investigation of the impact of bromodomain inhibition on cytoskeleton stability and contraction.

doi: 10.1186/s12964-024-01553-6

Figure Lengend Snippet: Fig. 6 In vitro functional validation of JQ1 attenuation of PDGF and TGFB stimulated contraction in pHBSMC. A Representative images of pHBSMC stimulated with PBS (Veh), PDGF, TGFB with DMSO or JQ1 for 16 h and stained with Vimentin, Phalloidin and DAPI. B Phalloidin intensity and cell shape was quantified in ImageJ. C RBMC were plated on 1.2 mg/mL collagen gels in a 12well plate in 1 mL of serum free media and incubated for 16 h with PBS (Veh), PDGF, TGFB with DMSO or JQ1. Select wells were stimulated for 30 min with a ROCK inhibitor as a negative control for contraction. Collage gels were separated from the walls of the wells and Imaged after 1.5 h to capture spontaneous (unstimulated contraction, followed by an additional 1.5 h in 5% FBS to facilitate contraction. D Contraction was quantified using ImageJ and measured as a percent of the changed area of the gel from the baseline. Data are representative of 3 independent trials. 1NC_FBS refers to negative control for FBS in which cells never receive FBS from platting to harvesting. 2NC_iROCK refers to the negative control for contraction in which cells are stimulated with a ROCK inhibitor for 30 min prior to observing contraction. *, p < 0.05, ** p < 0.01, ***, p < 0.001, ****, p < 0.0001 compared to control. #, p < 0.05, ## p < 0.01, ###, p < 0.001, ###, p < 0.0001 compared to PDGF. $, p < 0.05, $$ p < 0.01, $$$, p < 0.001, $$$$, p < 0.0001 compared to TGFB. Statistical significance was calculated with student ttest

Article Snippet: Validation of in silico predicted JQ1 sensitive genes via immunoblots Targets assessed via immunoblot included PDGFRβ, PDGFRα, ITGB1, ITGA5, αACTN, SRC, and CFL (Cell Signaling Technology).

Techniques: In Vitro, Functional Assay, Biomarker Discovery, Staining, Incubation, Negative Control, Control

Journal: Cell communication and signaling : CCS

Article Title: Investigation of the impact of bromodomain inhibition on cytoskeleton stability and contraction.

doi: 10.1186/s12964-024-01553-6

Figure Lengend Snippet: Fig. 7 Inhibition of MYC–MAX dimerization destabilizes the cytoskeleton and reduces contractility similar to JQ1. RBMC were plated in 4 well chamber slides and incubated with DMSO, JQ1, MYC-MAX dimerization inhibitors 10,048-F4 (F4), or 10,074-G5 (G5) for 16 h. Cells were stained for αSMA, Phalloidin or DAPI. Forty images were taken and stitched together with ImageJ to make a 5 × 8 field of view. B Phalloidin and αSMA intensity were quantified for each cell using ImageJ and plotted for each condition. C Cells were plated on collagen gels and stimulated for 16 h with DMSO, JQ1, F4 or G5 in serum free media and spontaneous contraction of gels was captured for 1.5 h prior to adding FBS to a final concentration of 5% to elicit contraction. D Contraction was quantified as a percentage in the change in area of the gel compared to baseline. E RNA was harvested from cells stimulated with DMSO, JQ1, F4 and G5 for 16 h and predicted JQ1 sensitive cytoskeleton associated genes was measured with qPCR

Article Snippet: Validation of in silico predicted JQ1 sensitive genes via immunoblots Targets assessed via immunoblot included PDGFRβ, PDGFRα, ITGB1, ITGA5, αACTN, SRC, and CFL (Cell Signaling Technology).

Techniques: Inhibition, Incubation, Staining, Concentration Assay

Journal: Nature Methods

Article Title: Single-cell EpiChem jointly measures drug–chromatin binding and multimodal epigenome

doi: 10.1038/s41592-024-02360-0

Figure Lengend Snippet: a , Schematic of single-cell EpiChem design. b , Track view displaying JQ1-btn signals in K562 and HGC27 cells at the representative loci. Genomic tracks of JQ1-btn binding in bulk level (JQ1-btn), single cell aggregate (JQ1-btn agg) and randomly selected 100 single cells alongside those of related proteins were presented. Chem-map data were downloaded from GSE209713 . c , Scatter-plot of the human–mouse species mixing test using JQ1-btn signals. Points are colored by the cell identity as human (red, >95% of reads mapping to hg19) and mouse (blue, >95% of reads mapping to mm10) or collision (gray, 5% to 95% of reads mapping to either genome). d , UMAP visualization showing K562 ( n = 2,000) and HGC27 cells ( n = 2,000) colored by cell types, based on JQ1-btn signals. e , Track view displaying THZ1-btn signals in K562 and HGC27 cells at the representative loci. Genomic tracks of small-molecule genomic bindings at bulk level (THZ1-btn), single cell aggregate (THZ1-btn agg) and randomly selected 100 single cells alongside those of related proteins were presented. f , Violin plot showing non-duplicated reads per cell of JQ1-btn and THZ1-btn of K562 ( n = 2,000), HGC27 ( n = 2,000) and mES cells ( n = 2,000). Numbers on the top of violin plot indicate the median value. g , Ridge plot showing the FRiP of JQ1-btn, THZ1-btn in K562, HGC27 and mES cells.

Article Snippet: JQ1-btn (HY-145667, CAS: 1635437-52-3) and THZ1-btn (HY-128867, CAS: 1604811-14-4) were obtained from MedChemExpress , .

Techniques: Single Cell, Binding Assay

Journal: Nature Methods

Article Title: Single-cell EpiChem jointly measures drug–chromatin binding and multimodal epigenome

doi: 10.1038/s41592-024-02360-0

Figure Lengend Snippet: a 1 H NMR spectrum (400 MHz) of Doxorubicin-btn (Dox-btn) in DMSO solvent. b Mass spectrum of Dox-btn. c-e Effect of different native small molecules and biotinylated derivatives on K562 (top), HGC27 (middle) and colorectal cancer organoids (bottom) proliferation. Cells were treated with varying concentrations of JQ1 and JQ1-btn ( c ), THZ1 and THZ1-btn ( d ), and Dox and Dox-btn ( e ). Mean ± s.d. (n = 3). f-h Confocal microscopy of K562 (top), HGC27 (middle) and colorectal cancer organoids (bottom) treated with different small molecules and biotinylated derivatives. Cells were stained for JQ1 and JQ1-btn ( f ), THZ1 and THZ1-btn ( g ), and Dox and Dox-btn ( h ) by the anti-biotin antibody. Nuclei were stained with DAPI. Scale bars, 20 μm ( f , g , h ). Shown are one of experiments repeated at least three times.

Article Snippet: JQ1-btn (HY-145667, CAS: 1635437-52-3) and THZ1-btn (HY-128867, CAS: 1604811-14-4) were obtained from MedChemExpress , .

Techniques: Solvent, Confocal Microscopy, Staining

Journal: Nature Methods

Article Title: Single-cell EpiChem jointly measures drug–chromatin binding and multimodal epigenome

doi: 10.1038/s41592-024-02360-0

Figure Lengend Snippet: a EpiChem workflow. b Track view displaying EpiChem signals of JQ1-btn in K562 and HGC27 cells, on the representative loci of cell-type specific drug binding sites ( FERMT3 ). BRD4 ( in situ ChIP) and JQ1-btn ( in vitro and in vivo experiments) data were obtained in this work, alongside JQ1 and biotin as two negative controls. Chem-map data were downloaded from GSE209713 . c Heatmap showing EpiChem signals around Chem-map BRD4 peak regions (51,963). The rows were sorted by the descending signals of Chem-map BRD4 signals. d Genomic distributions of JQ1-btn peaks from EpiChem in vitro and in vivo experiments. Total peaks number: K562-JQ1-btn in vitro : 72,799; K562-JQ1-btn in vivo : 39,036; HGC27-JQ1-btn in vitro : 58,907; HGC27-JQ1-btn in vivo : 38,039. e Venn diagram showing peak overlap in ( d ) of JQ1-btn ( in vitro and in vivo ) in K562 and HGC27 cells. Overlap peak numbers: K562 JQ1-btn in vivo and in vitro : 15,627; HGC27 JQ1-btn in vivo and in vitro :33,289; K562 JQ1-btn in vitro and HGC27 JQ1-btn in vitro : 19,288; K562 JQ1-btn in vitro and HGC27 JQ1-btn in vitro : 22,872. f Spearman correlation of JQ1-btn ( in vitro and in vivo ) and BRD4 ( in situ ChIP) binding signals within the Chem-map BRD4 peak regions (51,963) in K562 and HGC27 cells. g Spearman correlation of JQ1-btn ( in vitro or in vivo ) or BRD4 binding signals in K562 and HGC27 cells, in 10 kb genome wide. h Average JQ1-btn ( in vitro and in vivo ), JQ1 and biotin signals in K562 cells were plotted at the 5 kb flanking regions around the peak center of BRD4 from Chem-map (51,963).

Article Snippet: JQ1-btn (HY-145667, CAS: 1635437-52-3) and THZ1-btn (HY-128867, CAS: 1604811-14-4) were obtained from MedChemExpress , .

Techniques: Binding Assay, In Situ, In Vitro, In Vivo, Genome Wide

Journal: Nature Methods

Article Title: Single-cell EpiChem jointly measures drug–chromatin binding and multimodal epigenome

doi: 10.1038/s41592-024-02360-0

Figure Lengend Snippet: a The structure of scEpiChem sequencing library. b The expected number of barcode combinations using different numbers of PAT barcode introduced during tagmentation. The arrow indicated the expected barcode combinations in our scEpiChem experiments. c Expected collision rate for different cells per experiment in scEpiChem. d Read distribution for human/mouse mix-species scEpiChem data. The red dashed line indicates 1,000 non-duplicated reads, and barcodes with less than 1,000 reads were excluded from further analyses. e The broken line plot shows collision rate in the human-mouse species mixing test by scEpiChem single modality (JQ1-btn) data. f Benchmarking the ability of clustering of scEpiChem data with different sequencing depth. Box plots show the number of ‘Mixed’ cells occupying non-ambiguous clusters defined exclusively by either K562 or HGC27 cells in 80%, 40%, 20% down sampled reads. g The cluster accuracy of scEpiChem varied with down sampling reads. The line graph illustrates the change in the cluster accuracy, defined exclusively by either K562 or HGC27 cells in 80%, 40%, 20% down sampling reads. Shaded error band shows the 95% confidence interval (mean ± 2 SEM). h The boxplots show the number of single-cell detected reads in bulk peaks with scEpiChem after down sampling the reads of the single modality data from 20% (left) to 80% (right) (n = 2,000). i Heatmap showing Pearson correlation of aggregated single-cell genome-wide signals of JQ1-btn and H3K27me3 of dual modalities scEpiChem data in 2,000 K562 cells, and JQ1-btn or BRD4 signals from Chem-map, or H3K27me3 signals from ENCODE data. j-k ROC curves for aggregate JQ1&BRD4&H3K27ac ( j ) and JQ1&BRD4&ATAC ( k ) tri-modality scEpiChem data in K562 cells. Values next to indicated group names are the area under the ROC (AUC). The aggregate BRD4 data serves as the gold standard for specificity analysis.

Article Snippet: JQ1-btn (HY-145667, CAS: 1635437-52-3) and THZ1-btn (HY-128867, CAS: 1604811-14-4) were obtained from MedChemExpress , .

Techniques: Sequencing, Sampling, Single Cell, Genome Wide

Journal: Nature Methods

Article Title: Single-cell EpiChem jointly measures drug–chromatin binding and multimodal epigenome

doi: 10.1038/s41592-024-02360-0

Figure Lengend Snippet: a , Schematic of single-cell EpiChem design for the joint assay. b , UMAP embedding of dual modalities of scEpiChem data for JQ1-btn and H3K27me3. Connecting lines represent the same cells in different modalities (3,619 cells in two biological replicates). c , Violin plot showing the median non-duplicated reads of JQ1-btn and H3K27me3 of K562 ( n = 2,000) and HGC27 cells ( n = 2,000). Boxes in the violin plots: center marks the median and edges of boxes define the 25th and 75th percentiles. d , Track view of aggregate single cells of scEpiChem (dual modalities) K562 data and random 100 single cells with bulk reference, at the DESI2 loci. Chem-map data were downloaded from GSE209713 ; ENCODE H3K27me3 were downloaded from GSE31755 . e , Visualization of single-cell ATAC, BRD4, JQ1-btn modality of scEpiChem tri-modality data in K562 cells ( n = 1,261). Normalized signal was calculated by JQ1-btn, BRD4 and ATAC read counts in 56,352 BRD4 peaks and then z -score normalized. f , Violin plots depicting the calculated Cramér’s V of association between JQ1-btn & BRD4 modalities ( n = 1,261, median 0.71), JQ1-btn & ATAC ( n = 1,261, median 0.65), JQ1-btn & H3K27me3 ( n = 3,619, median 0.07) and JQ1-btn & random ( n = 1,261, median 0.03). g , Track view of aggregated single cells of scEpiChem (tri-modalities) data and random 100 single cells with bulk reference from ENCODE at the DESI2 loci. ENCODE ATAC were downloaded from GSE90409 .

Article Snippet: JQ1-btn (HY-145667, CAS: 1635437-52-3) and THZ1-btn (HY-128867, CAS: 1604811-14-4) were obtained from MedChemExpress , .

Techniques: Single Cell

Journal: Nature Methods

Article Title: Single-cell EpiChem jointly measures drug–chromatin binding and multimodal epigenome

doi: 10.1038/s41592-024-02360-0

Figure Lengend Snippet: a Track view of JQ1-btn and BRD4 signals on the representative loci in K562 cells. JQ1-btn specific loci (left), BRD4 specific loci (middle) and JQ1-btn and BRD4 shared loci (right). b Venn diagram showing peak overlap of JQ1-btn and BRD4 in K562 cells. Overlap peak numbers of JQ1-btn and BRD4: 19,342; JQ1-btn specific peak numbers: 5,289; BRD4 specific peak numbers: 3,213. c Genomic distributions of scEpiChem peaks of JQ1-btn and BRD4 in K562 cells. d Heatmap showing JQ1 and BRD4 binding signals of scEpiChem data in JQ1-specific (top), shared (middle) and BRD4-specific (bottom) peaks in K562 cells.

Article Snippet: JQ1-btn (HY-145667, CAS: 1635437-52-3) and THZ1-btn (HY-128867, CAS: 1604811-14-4) were obtained from MedChemExpress , .

Techniques: Binding Assay

Journal: Nature Methods

Article Title: Single-cell EpiChem jointly measures drug–chromatin binding and multimodal epigenome

doi: 10.1038/s41592-024-02360-0

Figure Lengend Snippet: a Spearman correlation of THZ1-btn and Dox-btn binding signals in bulk and single cell data within the TSS 5±kb regions in human CRC organoids. b ROC curves for CRC bulk and single cell aggregated THZ1-btn (4,424 cells) or Dox-btn (4,793 cells), using CRC bulk THZ1-btn or Dox-btn data as gold standard. AUC: THZ1-btn, 0.9561; Dox-btn, 0.8361. c UMAP showing scEpiChem data (small molecules and H3K27ac) on CRC organoids (n = 14,027 cells) with projections of the gene activity scores of the EMT score and CDH1. The EMT score was calculated based on 1,184 signature genes ( http://www.dbemt.bioinfo-minzhao.org/ ). d UMAP showing the batch effect of scEpiChem (small molecules and ATAC; small molecules, target and ATAC) on CRC organoids data from two samples, p1201 (n = 6,209) and p20 (n = 3,222); p1201 (n = 12,790) and p20 (n = 8,993). e Track view of H3K27ac and Dox-btn signals on the representative loci in CRC organoid cells. f Genomic distributions of scEpiChem peaks of Dox-btn and H3K27ac in CRC organoid cells. g Venn diagram showing peak overlap of Dox-btn and H3K27ac in CRC organoid cells. h GO terms enriched by Dox-btn-specific peaks. P-value was calculated by binomial test. The results of GO term enrichment analysis using a hypergeometric test, with two-sided statistical tests and adjustments for multiple comparisons employing the Benjamini-Hochberg method. i-j Distribution of the median non-duplicated reads for single cells in H3K27ac (n = 12,066), Dox-btn (n = 3,052), JQ1-btn (n = 4,012) and THZ1-btn (n = 3,453) with scEpiChem (small molecules and H3K27ac) ( i ) and ATAC (n = 10,672), BRD4 (n = 5,809) and JQ1-btn (n = 5,302) with scEpiChem (small molecules, target and ATAC) ( j ) in CRC organoids data. Boxes in the violin plots: center marks the median; edges of boxes define the 25th and 75th percentiles.

Article Snippet: JQ1-btn (HY-145667, CAS: 1635437-52-3) and THZ1-btn (HY-128867, CAS: 1604811-14-4) were obtained from MedChemExpress , .

Techniques: Binding Assay, Single Cell, Activity Assay

Journal: Nature Methods

Article Title: Single-cell EpiChem jointly measures drug–chromatin binding and multimodal epigenome

doi: 10.1038/s41592-024-02360-0

Figure Lengend Snippet: a , UMAP showing scEpiChem (small molecules and H3K27ac) in human CRC organoids ( n = 14,027), identified as epithelial cells ( n = 9,341) and Intermediate EMT cells ( n = 4,686). b , UMAP showing undetected batch effects in different scEpiChem experiments (small molecules and H3K27ac) in CRC organoids ( n = 14,027), visualizing Dox-btn + H3K27ac ( n = 4,793), JQ1-btn + H3K27ac ( n = 4,810) and THZ1-btn + H3K27ac ( n = 4,424). c , Pseudotime trajectory showing EMT progression. d , Track view displaying signals of H3K27ac and three small molecules in epithelial and intermediate EMT cells on the representative loci of cell type-specific drug binding sites. The pink, blue and purple shading represents epithelial cell-specific, intermediate EMT-specific and common peaks, respectively. e , UMAP projections showing gene activity scores of GPN3 , DDX42 and STRADA among small molecules. f , Aggregate curves (left) and heatmaps (middle) showing dynamic genomic signals of H3K27ac and small molecules (Dox-btn, JQ1-btn and THZ1-btn) along the pseudotime. Representative genes in each cluster were labeled on the right. The top three enriched GO terms in each cluster are shown (right). De novo transcription factor motifs in peaks were discovered using Homer. P values were calculated by the Binomial test. The results of GO term enrichment analysis using a hypergeometric test, with two-sided statistical tests and adjustments for multiple comparisons employing the Benjamini–Hochberg method.

Article Snippet: JQ1-btn (HY-145667, CAS: 1635437-52-3) and THZ1-btn (HY-128867, CAS: 1604811-14-4) were obtained from MedChemExpress , .

Techniques: Binding Assay, Activity Assay, Labeling

Journal: Nature Methods

Article Title: Single-cell EpiChem jointly measures drug–chromatin binding and multimodal epigenome

doi: 10.1038/s41592-024-02360-0

Figure Lengend Snippet: a Track view of JQ1-btn and BRD4 signals on the representative loci in epithelial cells and Intermediate EMT. b Venn diagram showing peak overlap of JQ1-btn and BRD4 in epithelial cells and Intermediate EMT. Overlap peak numbers of JQ1-btn and BRD4 in epithelial cells (26,999) and Intermediate EMT (21,459); JQ1-btn specific peak numbers in epithelial cells (5,614) and Intermediate EMT (8,326); BRD4 specific peak numbers in epithelial cells (15,733) and Intermediate EMT (8,630). c Genomic distributions of scEpiChem peaks of JQ1-btn and BRD4 in epithelial cells and Intermediate EMT. d Heatmap showing JQ1 and BRD4 binding signals of scEpiChem data in JQ1-specific (top), shared (middle) and BRD4-specific (bottom) peaks in epithelial cells and Intermediate EMT.

Article Snippet: JQ1-btn (HY-145667, CAS: 1635437-52-3) and THZ1-btn (HY-128867, CAS: 1604811-14-4) were obtained from MedChemExpress , .

Techniques: Binding Assay

Journal: Nature Methods

Article Title: Single-cell EpiChem jointly measures drug–chromatin binding and multimodal epigenome

doi: 10.1038/s41592-024-02360-0

Figure Lengend Snippet: a , UMAP showing scEpiChem (JQ1-btn, BRD4 and ATAC) in human CRC organoids ( n = 19,983), identified as epithelial cells ( n = 10,981) and Intermediate EMT cells ( n = 9,004). b , The pseudotime trajectory of the EMT progression. c , UMAP projections showing the gene activity scores of VIM and CDH1 . d , Heatmaps showing dynamic genomic signals of BRD4 and JQ1 along the pseudotime. Rows were clustered by hierarchical co-clustering and smoothed by the step size of one. Representative genes in each cluster were labeled on the right. e , Top five enriched GO terms of each small molecule (C1:1,384, C2:3,615, C3:2,917) are shown on the right. The P values of GO term enrichment analysis were calculated using a hypergeometric test, with two-sided statistical tests and adjustments for multiple comparisons employing the Benjamini–Hochberg method. f , Violin plots showing Cramér’s V of association between JQ1-btn + BRD4 (median 0.69), JQ1-btn + ATAC (median 0.48), BRD4 + ATAC (median 0.51) in epithelial cells ( n = 10,981); and JQ1-btn + BRD4 (median 0.71), JQ1-btn + ATAC (median 0.50), BRD4 + ATAC (median 0.47) in Intermediate EMT cells ( n = 9,004) and JQ1-btn + random ( n = 1,261, median 0.03). The same number of simulated random genomic regions (42,782) as for BRD4 peaks was used.

Article Snippet: JQ1-btn (HY-145667, CAS: 1635437-52-3) and THZ1-btn (HY-128867, CAS: 1604811-14-4) were obtained from MedChemExpress , .

Techniques: Activity Assay, Labeling

Journal: Nature Methods

Article Title: Single-cell EpiChem jointly measures drug–chromatin binding and multimodal epigenome

doi: 10.1038/s41592-024-02360-0

Figure Lengend Snippet: a , Experimental workflow for small-molecule drug treatment of human CRC organoids. b , UMAP projections showing the gene activity scores of VIM , CDH1 . c , UMAP showing scEpiChem (JQ1-btn and H3K27ac) in human CRC organoids ( n = 8,797), identified as epithelial cells ( n = 6,334) and intermediate EMT cells ( n = 2,463). The stacked bar plot shows the proportion of different cell types at each time point (right). d , Heatmaps showing dynamic genomic signals of JQ1-btn along the pseudotime. Cells were ordered by 33,131 peaks in JQ1-btn with 229 columns in Day0 (untreated), 228 columns in Day3&5 (treated). The top three enriched GO terms in each cluster are shown (right). e , UMAP showing scEpiChem (THZ1-btn and H3K27ac) in human CRC organoids ( n = 9,574), identified as epithelial cells ( n = 6,675) and intermediate EMT cells ( n = 2,899). f , Heatmaps showing dynamic genomic signals of THZ1-btn along the pseudotime. Cells were ordered by 22,054 peaks in THZ1-btn with 171 columns in Day0 (untreated) and 193 columns in Day3&5 (treated). The top three enriched GO terms in each cluster were shown (right). g , UMAP showing scEpiChem (Dox-btn and H3K27ac) in human CRC organoids ( n = 9,239), identified as epithelial cells ( n = 6,318) and intermediate EMT cells with a high EMT score ( n = 2,921). h , Heatmaps showing dynamic genomic signals of Dox-btn along the pseudotime. Cells were ordered by 17,908 peaks in Dox-btn with 174 columns in Day0 (untreated) and 282 columns in Day3&5 (treated, columns referring to metacells with 50 single cells each). The top three enriched GO terms in each cluster were shown. The top three enriched GO terms in each cluster are shown. P values of GO term enrichment analysis in d , f and h were calculated using hypergeometric test, with two-sided statistical tests and adjustments for multiple comparisons employing the Benjamini–Hochberg method.

Article Snippet: JQ1-btn (HY-145667, CAS: 1635437-52-3) and THZ1-btn (HY-128867, CAS: 1604811-14-4) were obtained from MedChemExpress , .

Techniques: Activity Assay

Journal: Nature Methods

Article Title: Single-cell EpiChem jointly measures drug–chromatin binding and multimodal epigenome

doi: 10.1038/s41592-024-02360-0

Figure Lengend Snippet: a Heatmaps showing dynamic genomic signals of Dox-btn and H3K27ac along the pseudotime. Left, aggregate curves showing signals of Dox-btn and H3K27ac in Day0 and Day3&5 along the pseudotime, respectively. Middle, heatmaps showing dynamic genomic signals of Dox-btn along the pseudotime. Cells were ordered by 17,908 peaks in Dox-btn with 174 columns in Day0 (untreated) and 282 columns in Day3&5 (treated, columns referring to metacells, 50 single cells each). Rows were clustered by hierarchical co-clustering and smoothed by the step size of one. The binding dynamics of the signals (peaks with less than 10 reads were removed) of JQ1-btn was presented along the EMT progression. Right, Top 3 enriched GO terms in each cluster were shown. De novo TF motifs in peaks were discovered using Homer. P value was calculated by the Binomial test. The results of Gene Ontology (GO) term enrichment analysis using a hypergeometric test, with two-sided statistical tests and adjustments for multiple comparisons employing the Benjamini-Hochberg method. b-c ChromVAR identifying TF dynamics during EMT in differential regions between Dox-btn and H3K27ac for Day3&5 CRC samples based on the signals in C1 peaks as in ( a ). Among the dynamic TF genes with increasing TF activity score along pseudotime (44 genes in Dox-btn, 43 genes in H3K27ac, FDR < 0.001), only 7 TFs are shared; Among the dynamic TF genes with decreasing TF activity score along pseudotime (46 genes in Dox-btn, 40 genes in H3K27ac, FDR < 0.001), only 4 TFs are shared.

Article Snippet: JQ1-btn (HY-145667, CAS: 1635437-52-3) and THZ1-btn (HY-128867, CAS: 1604811-14-4) were obtained from MedChemExpress , .

Techniques: Binding Assay, Activity Assay

Journal: eLife

Article Title: Quantitative mapping of proteasome interactomes and substrates using ProteasomeID

doi: 10.7554/eLife.93256

Figure Lengend Snippet: ( a ) Immunoblot of miniTurbo fusion proteins performed on lysates collected from HEK293T cells stably transfected with PSMA4-miniTurbo-FLAG or miniTurbo-FLAG following 4 days of incubation with (+tet) or without (−tet) tetracycline. Immunoblot against GAPDH was used as loading control. ( b ) Streptavidin-HRP immunoblot following induction of miniTurbo fusion proteins with tetracycline and supplementation of biotin for 2 hr. Immunoblot against GAPDH was used as loading control. ( c ) Streptavidin-HRP immunoblot following induction of miniTurbo fusion proteins with tetracycline and supplementation of biotin for indicated times. For the sample used as negative control biotin supplementation was omitted (-biotin). Ponceau staining was used as loading control. Bar plots on the left depict densitometric quantification of the immunoblot. Samples were normalized to the band intensity of PSMA4-BirA* sample not supplemented with biotin (untreated sample). ( d ) MA plot of proteins enriched by streptavidin pull-down and analyzed by DIA mass spectrometry from PSMA4-miniTurbo and miniTurbo control cell lines. Data were obtained from n=4 biological replicates. ( e ) Comparison of log2 fold changes for streptavidin-enriched proteins from PSMA4-BirA* and PSMA4-miniTurbo compared to their respective controls. Proteins significant (Q value <0.05) and displaying a log2 fold change >0 in both comparisons were considered for the analysis. ( f ) ROC analysis of the classifier used to define ProteasomeID (PSMA4-miniTurbo) enriched proteins. ( g ) Distribution of enrichment scores for PSMA4-miniTurbo enriched proteins. Calculated by the classifier algorithm for proteasome subunits (set of true positives) and mitochondrial matrix proteins (set of true negatives). The dashed vertical line indicates the enrichment score cut-off to define ProteasomeID enriched proteins at FPR <0.05. ( h ) Immunoblot for K48 ubiquitylated proteins from PSMA4-miniTurbo cells treated with 20 µM MG132 for 4 h. As a negative control the same cell line was treated in the same way with DMSO only. Ponceau staining was used as loading control. ( i ) Principal component analysis (PCA) of ProteasomeID data obtained from cell lines expressing PSMA4-miniTurbo and control (miniTurbo), and PSMA4-miniTurbo following exposure to proteasome inhibitor MG132. The smaller dots represent individual samples and the larger dots the centroids of each group. Ellipses represent 95% confidence intervals. The percentage of variance explained by the first two principal components (PC) axes is reported in the axis titles. n=4, biological replicates. ( j ) Cycloheximide-chase experiment on stability of 3 potential novel proteasome substrate proteins. PSMA4-BirA*cells were incubated with 50 μg/ml cycloheximide (CHX) for the indicated times in the presence or absence of MG132 (20 μM) and tetracycline (1 µg/µl). Cell lysates were then prepared for western blot analysis of steady-state levels of c-Myc, ARMC6, and BRAT1 and TIGD5. c-Myc was used as a positive control as it is a well known proteasome substrate. Tet = tetracycline, CHX = cycloheximide. ( k ) Principal component analysis (PCA) of ProteasomeID data obtained from cells expressing PSMA4-miniTurbo exposed to the proteasome inhibitor MG132 and/or the PROTAC KB02-JQ1. The smaller dots represent individual samples and the larger dots the centroids of each group. Ellipses represent 95% confidence intervals. The percentage of variance explained by the first two principal components (PC) axes is reported in the axis titles. n=4, biological replicates. Figure 4—figure supplement 1—source data 1. Raw unedited gels for . Figure 4—figure supplement 1—source data 2. Uncropped and labeled gels for .

Article Snippet: Peptide, recombinant protein , KB02-JQ1 , MedChemExpress , HY-129917 , .

Techniques: Western Blot, Stable Transfection, Transfection, Incubation, Control, Negative Control, Staining, Mass Spectrometry, Comparison, Expressing, Positive Control, Labeling

Journal: eLife

Article Title: Quantitative mapping of proteasome interactomes and substrates using ProteasomeID

doi: 10.7554/eLife.93256

Figure Lengend Snippet: ( a ) Scheme of ProteasomeID workflow in HEK293T cells including proteasome inhibition by MG132 and treatment with PROTAC KB02-JQ1. The experimental design is analogous to the one depicted in with the additional PROTAC treatment achieved by addition of 10 µM KB02-JQ1 12 hr before cell harvesting. D: day; h: hour; Tet: tetracycline; Bio: biotin. ( b ) Volcano plot of proteins enriched by streptavidin pull-down and analyzed by DIA mass spectrometry from PSMA4-miniTurbo cells treated with KB02-JQ1 PROTAC molecule (P) and PSMA4-miniTurbo cells treated with both PROTAC molecule (P) and MG132 proteasome inhibitor (I). Cut offs for enriched proteins: log2 fold change >1 and Q value <0.05. n=4, biological replicates. Enrichment of BRD containing proteins is highlighted in violet boxes. ( c ) Bar plots comparing the levels of BRD-containing proteins following streptavidin enrichment from PSMA4-miniTurbo expressing cells exposed to the proteasome inhibitor MG132 and/or the PROTAC KB02-JQ1. mT: miniTurbo control cell line; A4-mT: PSMA4-miniTurbo cell line; I: proteasome inhibition by MG132; P: PROTAC (KB02-JQ1). Data are shown as mean ± standard error from n=4 biological replicates.

Article Snippet: Peptide, recombinant protein , KB02-JQ1 , MedChemExpress , HY-129917 , .

Techniques: Inhibition, Cell Harvesting, Mass Spectrometry, Expressing, Control