Journal: Oncotarget

Article Title: BH3-mimetics and BET-inhibitors elicit enhanced lethality in malignant glioma

doi: 10.18632/oncotarget.16365

Figure Lengend Snippet: A .- C . U87MG, LN229, T98G established glioblastoma cell lines, GBM39, GBM6 and GBM14 patient-derived xenograft cultures and NCH644 and NCH421k stem cell-like glioma cultures were treated as indicated with JQ1. After 72h of treatment, CellTiter-Glo assays were performed. IC 50 values were calculated. Column: mean. Error bar: standard deviation (SD). n = 3. D ., E ., U87MG, LN229, T98G established glioblastoma cell lines, GBM39, GBM6 and GBM14 patient-derived xenograft cultures were treated with ABT263, JQ1 or the combination of both. After 72h of treatment, CellTiter-Glo assays were performed. Column: mean. Error bar: standard deviation (SD). n = 3. Statistical analysis was performed and p values were calculated. A p-value of less than 0.05 was considered statistically significant. F .- H ., LN229, T98G and NCH644 glioblastoma cells were treated for 72 hours with ABT263, JQ1 or the combination and analyzed by CellTiter-Glo assay. CI values and fraction affected were calculated using the CompuSyn software (ComboSyn, Inc., Paramus, NJ, U.S.A.). Data points located below 1 (CI value less than 1) indicate a synergistic drug-drug interaction and data points larger than 1 indicate an antagonistic drug-drug interaction. Some data points overlap and are therefore not represented on the graphical chart. A colored line highlights CI value 1. For individual values, please refer to Table .

Article Snippet: Notably, stem cell-like glioma cells display a remarkable sensitivity to the drug combination of BET-inhibitors, such as JQ1 and OTX015, and BH3-mimetics.

Techniques: Derivative Assay, Standard Deviation, Glo Assay, Software

Journal: Oncotarget

Article Title: BH3-mimetics and BET-inhibitors elicit enhanced lethality in malignant glioma

doi: 10.18632/oncotarget.16365

Figure Lengend Snippet: CI values for glioblastoma cultures after combinatorial treatments with ABT263 and JQ1

Article Snippet: Notably, stem cell-like glioma cells display a remarkable sensitivity to the drug combination of BET-inhibitors, such as JQ1 and OTX015, and BH3-mimetics.

Techniques:

Journal: Oncotarget

Article Title: BH3-mimetics and BET-inhibitors elicit enhanced lethality in malignant glioma

doi: 10.18632/oncotarget.16365

Figure Lengend Snippet: A ., B ., C . representative histograms of LN229, U87 and T98G glioblastoma cells that were treated for the indicated time points as indicated with JQ1, ABT263 or both prior to staining with propidium iodide and flow cytometric analysis. D .- F ., LN229, U87 and T98G glioblastoma cells were treated for the indicated time points as indicated with OTX015, Obatoclax or the combination. G ., representative histograms of T98G glioblastoma cells treated with ABT263, JQ1 or the combination as indicated for 48h prior to staining for annexin V and propidium iodide and flow cytometric analysis. H ., representative histograms of T98G glioblastoma cells that were treated with JQ1, ABT263, or both prior to staining with TMRE and flow cytometric analysis. I ., T98G glioblastoma cells were treated for 24 h with JQ1, ABT263 or the combination. Whole-cell extracts were examined by Western blot analysis for PARP, cleaved caspase 9 (C9) and cleaved caspase 3. Vinculin Western blot analysis was performed to confirm equal protein loading.

Article Snippet: Notably, stem cell-like glioma cells display a remarkable sensitivity to the drug combination of BET-inhibitors, such as JQ1 and OTX015, and BH3-mimetics.

Techniques: Staining, Western Blot

Journal: Oncotarget

Article Title: BH3-mimetics and BET-inhibitors elicit enhanced lethality in malignant glioma

doi: 10.18632/oncotarget.16365

Figure Lengend Snippet: A ., Established glioblastoma cells, U87,LN229, T98G and stem cell-like glioma cells, NCH644 were treated with increasing concentrations of JQ1 as indicated for 72h. Whole cell extracts were collected and Western blot analysis was performed for Usp9X, Mcl-1, Bcl-2, Bcl-xL, Noxa and Bim. Actin served as loading control. B ., C . U87, LN229 and T98G cells were treated with ABT263, JQ1 or the combination of both for 24h (B) and 48h (C). Whole cell extracts were collected and Western blot analysis was performed for Mcl-1, Bcl-2, Bcl-xL, Noxa and Bim. Actin served as loading control. D ., U87 and LN229 cells were treated with ABT263, JQ1 or the combination of both. After 7h cells were harvested and RNA was isolated. Real-time PCR for Noxa was conducted and the results were normalized to 18S. Column: mean. Error bar: standard error of measurement (SEM). G, LN229 cells were transfected with non-targeting (n.t.) siRNA or an ATF4 specific siRNA. 72h after transfection cells were treated with ABT263 and JQ1 for 7h. Cells were harvested and analyzed by western blotting for ATF4 and Noxa. Actin serves as loading control.

Article Snippet: Notably, stem cell-like glioma cells display a remarkable sensitivity to the drug combination of BET-inhibitors, such as JQ1 and OTX015, and BH3-mimetics.

Techniques: Western Blot, Control, Isolation, Real-time Polymerase Chain Reaction, Transfection

Journal: Oncotarget

Article Title: BH3-mimetics and BET-inhibitors elicit enhanced lethality in malignant glioma

doi: 10.18632/oncotarget.16365

Figure Lengend Snippet: A .- D ., Representative flow plots of LN229 cells that were treated with n.t.-siRNA or 2 different Bcl-xL siRNAs prior to additional treatment with either solvent or JQ1. Staining for propidium iodide and flowcytometric analysis was performed to determine the fraction of subG1 cells. The results were quantified (D). Knockdown of Bcl-xL was confirmed by Western Blot analysis (C). Actin served as loading control (C). E .- G . LN229 cells were treated with n.t.-siRNA or Noxa-siRNA or Bak-siRNA prior to treatment with solvent or the combination of 1μM ABT263 and 5μM JQ1 as indicated for 24 h. Staining for propidium iodide and flow cytometric analysis was performed to determine the fraction of subG1 cells. Representative flow plots are shown (E). The quantifications are shown in (G). Noxa and Bak knockdowns were confirmed by Western blot analysis (F). H ., LN229 cells were transfected with n.t.-siRNA or four individual Mcl-1 siRNAs for 48h. Subsequently, cells were treated with ABT263 and analyzed by CellTiter-Glo assay. The concentrations for ABT263 are in μM. Column: mean. Error bar: standard deviation (SD). I ., LN229 cells were transfected as in G and analyzed for protein expression of Mcl-1. Actin served as a loading control.

Article Snippet: Notably, stem cell-like glioma cells display a remarkable sensitivity to the drug combination of BET-inhibitors, such as JQ1 and OTX015, and BH3-mimetics.

Techniques: Solvent, Staining, Knockdown, Western Blot, Control, Transfection, Glo Assay, Standard Deviation, Expressing

Journal: Oncotarget

Article Title: BH3-mimetics and BET-inhibitors elicit enhanced lethality in malignant glioma

doi: 10.18632/oncotarget.16365

Figure Lengend Snippet: A ., LN229 cells were transfected with n.t.-siRNA or c-myc-siRNA-1 prior to treatment with solvent or ABT263 as indicated for 24 h. Staining for propidium iodide and flow cytometric analysis were performed to determine the fraction of subG1 cells. Representative flow plots are shown. The quantifications are shown in C . c-myc knockdown was confirmed by Western blot analysis B . D ., LN229 cells were transfected with n.t.-siRNA or c-myc-siRNA-2 prior to treatment with solvent or ABT263 as indicated for 24 h. Staining for propidium iodide and flow cytometric analysis were performed to determine the fraction of subG1 cells. Representative flow plots are shown. The quantifications are shown in F . c-myc knockdown was confirmed by Western blot analysis E . G . U87, T98G, LN229 (established glioblastoma cells), NCH644 (stem cell-like glioma cells) and GBM6 (patient-derived xenograft) cells were treated with JQ1 for 72 hours. Protein extracts were prepared and samples were analyzed by conventional western blot analysis for the expression of c-myc. Actin serves as a loading control. Multiple exposures are presented due to the fact that cells express different baseline levels of c-myc. * indicate different exposure times for c-myc protein.

Article Snippet: Notably, stem cell-like glioma cells display a remarkable sensitivity to the drug combination of BET-inhibitors, such as JQ1 and OTX015, and BH3-mimetics.

Techniques: Transfection, Solvent, Staining, Knockdown, Western Blot, Derivative Assay, Expressing, Control

Journal: Nature Communications

Article Title: Enhancer and super-enhancer dynamics in repair after ischemic acute kidney injury

doi: 10.1038/s41467-020-17205-5

Figure Lengend Snippet: a C57BL/6N mice (10- to 12-week-old males) were treated daily starting at the day of SHAM or IRI surgery (d0) with JQ1 (50 mg/kg BW) or vehicle (DMSO/10% ß-cyclo dextrin 1:10). b Serum creatinine (mg/dL) values in SHAM and IRI animals with vehicle or JQ1 at day 1 after surgery are shown (individual data points and mean ± SD). SHAM: n = 4; IRI: n = 12 biologically independent samples. c Survival curves after IRI surgery (ischemic time was 26 min at 37 °C). JQ1 treatment leads to 80% mortality after IRI surgery. Table shows number of animals alive at indicated days. d Experimental design of delayed treatment with JQ1 (50 mg/kg BW) after IRI starting at day 1, day 2, day 3 or day 4. e Serum creatinine levels at day 1 after IRI, verifying that AKI was induced to a roughly equivalent extent in each of the groups of animals that were subsequently treated with JQ1 (individual data points and mean ± SD). IRI veh: n = 11; IRI d1: n = 12; IRI d2: n = 8; IRI d3: n = 9; IRI d4: n = 7 biologically independent samples. f Survival curve after delayed JQ1 treatment starting day 1, day 2, day 3 or day 4. Table indicates number of animals alive at each day. Box indicates start of vehicle or JQ1 treatment. Source data are provided as a file.

Article Snippet: The BET inhibitor JQ1 or vehicle was provided to us by Dr. Jay Bradner (formerly at Brigham and Women’s Hospital now at the Novartis Institutes for Biomedical Research).

Techniques:

Journal: Nature Communications

Article Title: Enhancer and super-enhancer dynamics in repair after ischemic acute kidney injury

doi: 10.1038/s41467-020-17205-5

Figure Lengend Snippet: a Principal component (PC) analysis of normalized RNA-seq data matrix of SHAM, IRI and IRI JQ1 kidney cortex samples on day 2 after IRI. b Serum creatinine values at days 0, 1, and 2 after IRI comparing SHAM, IRI and IRI JQ1-treated groups. c Genes significantly differentially regulated between SHAM and IRI are shown in a heatmap of SHAM, IRI, and IRI JQ1; JQ1 leads to suppression of ~40% upregulated genes after injury. d Comparison of significantly upregulated genes 2 days after injury (SHAM vs. IRI up) and significantly down-regulated genes after JQ1 treatment (IRI vs IRI JQ1 down) with an overlap of 718 transcripts (Chi-square test P < 0.001) shown in a Venn diagram. e KEGG pathways enriched for the 718 genes upregulated after injury and down-regulated by JQ1. f Top 30 upregulated genes after injury shown in a heatmap of SHAM, IRI and IRI JQ1 samples. g Representative KIM-1/Ki67-immunostained IRI kidney cortex treated with vehicle or JQ1 at day 2 after injury, Quantification of Ki67+ cells (Ki67+ cells/total number of cells (DAPI)) and KIM-1+ surface area per hpf ( n = 4, 7 high power fields (hpf) per sample). Scale bar: 50 μm. h Fold change of Mki67 and KIM-1 after IRI comparing vehicle and JQ1 treated animals at day 2 after injury (vehicle: n = 4, JQ1: n = 6). i Genome-wide assessment of Pol II binding: Genome-wide coverage blots of Pol II on the gene body. Pol II binding after JQ1 treatment is increased at the TSS and decreased across the gene body indicating Pol II pausing j Pol II ChIP-seq tracks at the Spp1 gene body. k Fold change of Spp1 after IRI comparing vehicle and JQ1 treated animals at day 2 after injury. n = 4. Data represent the mean ± min, max. Box contains 50% of the data. t -test (two-sided) ( g , h , k ). Source data are provided as a file.

Article Snippet: The BET inhibitor JQ1 or vehicle was provided to us by Dr. Jay Bradner (formerly at Brigham and Women’s Hospital now at the Novartis Institutes for Biomedical Research).

Techniques: RNA Sequencing, Comparison, Genome Wide, Binding Assay, ChIP-sequencing

Journal: Nature Communications

Article Title: Enhancer and super-enhancer dynamics in repair after ischemic acute kidney injury

doi: 10.1038/s41467-020-17205-5

Figure Lengend Snippet: a BALB/c mice (10- to 12-week-old males) were treated daily with JQ1 (50 mg/kg) or vehicle (DMSO/10% ß-cyclo dextrin 1:10) starting on the day of aristolochic acid (AA) injection (3 mg/kg BW) or day 7 after AA injection. Mice were sacrificed on day 21 after AA injection. b Serum creatinine (mg/dL) trajectories of vehicle and JQ1 treated mice from day 0 until day 21 after AA (mean ± SD). vehicle: n = 7; JQ1 d0: n = 6; JQ1 d7: n = 8 biologically independent samples. c Survival curves after AA injection: 100% survial in vehicle and JQ1 d7 group, 67% survival in mice treated with JQ1 from day 0 until day 21 after AA injection. d Representative Ki67-1/Acta2-immunostained AAN kidneys treated with vehicle or JQ1 d0 or JQ1 d7. e Quantification of α-SMA+ (Acta2) surface area. Percentage of Ki67+ cells (Ki67+ cells/total number of cells (DAPI) per hpf). vehicle: n = 7 (60 hpf); JQ1 d0: n = 4 (33 hpf); JQ1 d7: n = 8 (64 hpf) at least 8 hpf per sample. f C57BL/6N mice (8- to 10-week-old males) were treated daily starting at the day of UUO surgery with JQ1 (50 mg/kg) or vehicle, and were sacrificed on day 10 after surgery. g Representative trichrome-stained and Sirius-red stained sections. h Quantification of fibrotic area (masson trichrome +-stained) ( n = 7, 5 hpf per sample) and Sirius red+ area (collagen) ( n = 7, at least 7 hpf per sample. t -test (two-sided). Data represent the mean ± min, max. Box contains 50% of the data. Scale bars: 100 µm ( d ), 50 μm ( g ). Source data are provided as a file.

Article Snippet: The BET inhibitor JQ1 or vehicle was provided to us by Dr. Jay Bradner (formerly at Brigham and Women’s Hospital now at the Novartis Institutes for Biomedical Research).

Techniques: Injection, Staining

Journal: Cancer Medicine

Article Title: Development of a novel cell line‐derived xenograft model of primary herpesvirus 8‐unrelated effusion large B‐cell lymphoma and antitumor activity of birabresib in vitro and in vivo

doi: 10.1002/cam4.4394

Figure Lengend Snippet: BET inhibitors suppress cell growth and induce cell cycle arrest and apoptosis in vitro . (A) Cell growth assays of Pell‐1 and HHV8‐positive PEL cell lines (BCBL‐1, JSC‐1, BC‐3, and KS‐1) were conducted 48 h after treatment with the indicated doses of JQ1 or birabresib. The numbers of viable cells are normalized to a percentage of the viable cell numbers of vehicle (DMSO)‐treated controls. Pell‐1 cells were significantly more sensitive to JQ1 and birabresib than all PEL cell lines tested at concentrations of 0.5 μM and 5 μM, respectively. (B) Cell cycle analysis was conducted 24 h after treatment with JQ1 (0.5 μM) or birabresib (5 μM). Percentages of the cell population in each stage of the cell cycle are presented outside the graph. (C) Apoptosis assay was conducted 48 h after treatment with the indicated doses of JQ1 or birabresib. The graphs show the percentages of apoptotic cells in the total cell population. All experiments were performed three times, and data are expressed as the mean ± SEM. Significant differences are shown as * p < 0.05, ** p < 0.01

Article Snippet: The BET inhibitors JQ1 and birabresib (MK‐8628/OTX015) were purchased from Merck KGaA (Darmstadt, Germany) and MedChemExpress (Monmouth Junction, NJ, USA), respectively, and dissolved in dimethyl sulfoxide (DMSO).

Techniques: In Vitro, Cell Cycle Assay, Apoptosis Assay

Journal: Science Advances

Article Title: Synthetic lethal combination targeting BET uncovered intrinsic susceptibility of TNBC to ferroptosis

doi: 10.1126/sciadv.aba8968

Figure Lengend Snippet: ( A ) Effects of BET and CXCR2 antagonists on cell viability. The indicated BL1/2 (blue) and M/MSL (green) TNBC cell lines and MCF7 (luminal) and MCF10A (normal-like) lines were treated with the indicated doses of the drugs for 72 hours and stained with crystal violet. Representative pictures of reproducible effects from two to three independent experiments are shown. ( B , E , and F ) Dose-response curves of single agents and drug combinations in the indicated cell lines treated with varying concentrations of JQ1 and SB225022 (B) or JQ1 and BTZ (E and F) for 72 hours. Dose-response curves are presented as means of four (B) or three (E and F) repeats. ( C and G ) CI was calculated by the CompuSyn software with the Chou-Talalay equation using multiple doses and response points. CI values for three different indicated FA are shown. ( D ) Effects of BET and proteasome inhibitors on cell viability were assessed by crystal violet staining as described in (A). ( H ) Effects of drug combinations on spheroid growth. Representative images and viability assay (CellTiter-Blue, bar graph) of day 15 4T1 spheroids ( n = 6 spheroids). Control untreated and treated spheroids with single agents at the indicated doses and with the drug combinations are shown. Scale bar, 100 μm.

Article Snippet: Proteasome inhibitor (BTZ) and BET inhibitor (JQ1) were purchased from AdooQ BioScience (CA, USA).

Techniques: Staining, Software, Viability Assay, Control

Journal: Science Advances

Article Title: Synthetic lethal combination targeting BET uncovered intrinsic susceptibility of TNBC to ferroptosis

doi: 10.1126/sciadv.aba8968

Figure Lengend Snippet: ( A and B ) Effects of BET and CXCR2 inhibition (A) or BET and proteasome inhibition (B) on PARP (A and B) and caspase-3 cleavage (A) in the indicated cell lines (blue, BL1; black, M/MSL). Staurosporine (150 nM, 16 hours) was used as a positive control. The cells were treated with low doses of JQ1 and either SB225022 (A) or BTZ (B) for 24 hours as described in Materials and Methods, lysed, and assessed by Western blot (WB) for the indicated proteins. ( C ) Ferroptosis inhibitors rescue cell death induced by JQ1 and BTZ combination. The indicated cell lines were pretreated with the indicated cell death inhibitors for 1 hour and then for an additional 72 hours in the absence or presence of JQ1 and BTZ (see Materials and Methods). Cell viability (CellTiter-Blue) is presented as percentage of untreated cells. The effects of the inhibitors on their cognate death pathways are shown in fig. S6C. Mean values of three experiments are shown (means ± SD; table S6). ( D ) Iron chelators rescued cell death induced by JQ1 and BTZ treatment. The indicated TNBC cell lines were treated with JQ1 and BTZ (see Materials and Methods) for 72 hours in the absence or presence of deferoxamine (DFO) (100 μM) or 2,2′-dipyridyl (10 μM), and cell viability was assessed by crystal violet staining. ( E ) Representative confocal images of the indicated TNBC cell lines and T47D cells stained with C11-BODIPY (10 μM) as described in Materials and Methods. The cells were treated with cumene hydroperoxide (CH) (100 μM, 3 hours) as a positive control, and either with JQ1 + SB225022 or with JQ1 and BTZ (see Materials and Methods) for 16 hours. Scale bar, 10 μm. ( F ) Assessment of lipid peroxidation in breast cancer cells in response to BET and proteasome inhibition. TNBC (gray) and non-TNBC (green) cell lines were incubated with JQ1 and BTZ (see Materials and Methods) for 16 hours or with CH (100 μM, 3 hours). Where indicated, glutathione (1 mM; see Materials and Methods) was applied. Lipid peroxidation is shown as the ratio between fluorescence emission at 510 nm (green) and 590 nm (red) (see Materials and Methods). Mean values of three experiments are shown (mean values ± SD; table S7).

Article Snippet: Proteasome inhibitor (BTZ) and BET inhibitor (JQ1) were purchased from AdooQ BioScience (CA, USA).

Techniques: Inhibition, Positive Control, Western Blot, Staining, Incubation, Fluorescence

Journal: Science Advances

Article Title: Synthetic lethal combination targeting BET uncovered intrinsic susceptibility of TNBC to ferroptosis

doi: 10.1126/sciadv.aba8968

Figure Lengend Snippet: ( A and B ) Inhibition of BET and the proteasome increased iron levels (A) and decreased GSH levels (B) in TNBC cell lines. TNBC and non-TNBC (MCF7, SKBR3, T47D, and BT474) cells were incubated with JQ1 and BTZ (see Materials and Methods) for 16 hours, and total iron (A) and reduced GSH (B) levels were measured as described in Materials and Methods. Mean values of three experiments are shown (mean values ± SD; table S8). ( C ) Inhibition of BET and the proteasome increased ROS in TNBC cells. The indicated breast cancer cells were treated with JQ1 and BTZ (see Materials and Methods) for 12 hours or with tert -butyl hydrogen peroxide (TBHP; 100 μM) for 2 hours. Where indicated, GSH (1 mM) was applied 1 hour before drug treatment. Cells were then incubated with CM-H 2 DCFDA to measure ROS as described in Materials and Methods. Results are expressed as folds of control in at least three experiments (mean values ± SD; table S9).

Article Snippet: Proteasome inhibitor (BTZ) and BET inhibitor (JQ1) were purchased from AdooQ BioScience (CA, USA).

Techniques: Inhibition, Incubation, Control

Journal: Science Advances

Article Title: Synthetic lethal combination targeting BET uncovered intrinsic susceptibility of TNBC to ferroptosis

doi: 10.1126/sciadv.aba8968

Figure Lengend Snippet: ( A ) Box plot showing the expression of GPX4 in patients with breast cancer grouped by PAM50. The differences between the BL patients and any other PAM50 groups are significant ( t test, P < 0.001). ( B and C ) Effects of BET and proteasome inhibition on the levels of GPX4 protein (B) and transcript (C). The indicated TNBC cell lines were treated with JQ1, BTZ, or both (see Materials and Methods) for 24 hours. Levels of GPX4 protein were assessed by WB. Intensities of GPX4 bands were quantified, normalized, and presented as fold of control in the bar graph (B). GPX4 mRNA levels were assessed by qPCR. Mean values ± SD of at least two repeats are shown (C). ( D ) Effects of BET and proteasome inhibition on level of GPX4 protein and transcript in 4T1 tumors. Representative IHC staining of 4T1 tumors treated with OTX015 (25 mg/kg per day, orally), BTZ (0.25 mg/kg, IP, every fourth day), or both to detect protein expression is shown. GPX4 mRNA levels were evaluated by qPCR. Results are mean values ± SD of at least six mice per group. ( E ) Effects of BET and proteasome inhibition on level of GPX4 transcript and additional key ferroptosis genes. The indicated TNBC cell lines were treated with JQ1 and BTZ (see Materials and Methods) for 24 hours, and mRNA levels of the indicated genes were assessed by qPCR. The results are reported as fold of control. Mean values of at least three independent experiments are shown (mean values ± SD; table S11).

Article Snippet: Proteasome inhibitor (BTZ) and BET inhibitor (JQ1) were purchased from AdooQ BioScience (CA, USA).

Techniques: Expressing, Inhibition, Control, Immunohistochemistry

Journal: Pharmaceuticals

Article Title: TRPM3-Induced Gene Transcription Is under Epigenetic Control

doi: 10.3390/ph15070846

Figure Lengend Snippet: Pharmacological inhibitors of epigenetic gene regulation. ( A ) Modular structure of the transcriptional coactivator and acetyltransferase CBP. The paralog p300 has a similar modular structure. Both proteins have a nuclear receptor interaction domain (NRID), two transcriptional adapter zinc finger domains (TAZ), a kinase inducible domain interacting domain (KIX) that binds CREB, the CREB-related protein ATF-1, c-Jun and other transcription factors, a bromodomain, a PHD-RING module, and a histone acetyltransferase (HAT) domain. The C-terminal TAZ domain binds c-Fos. ( B ) Chemical structure of the CBP/p300 catalytic inhibitor A485. ( C ) Modular structure of the BET proteins BRD3 and BRD4. BET proteins have two bromodomains (BD) as well as an extra-terminal (ET) domain that functions as a protein binding module for other chromatin-modifying proteins. BRD4 contains additionally a C-terminal domain (CTD) that functions as a binding site for P-TEFb (positive transcription elongation factor b). ( D ) Chemical structure of the pan-BET-inhibitor JQ1.

Article Snippet: The BET bromodomain inhibitor JQ1 (PubChem ID 46907787),(6 S )-4-(4-Chlorophenyl)-2,3,9-trimethyl-6 H -thieno [3,2- f ][1,2,4]triazolo [4,3- a ][1,4]diazepine-6-acetic acid 1,1-dimethylethyl ester) was purchased from Adooq Bioscience (Irvine, CA, USA, #A12729), dissolved in DMSO and used at a final concentration of 0.5 μM.

Techniques: Protein Binding, Binding Assay

Journal: Pharmaceuticals

Article Title: TRPM3-Induced Gene Transcription Is under Epigenetic Control

doi: 10.3390/ph15070846

Figure Lengend Snippet: Pharmacological inhibition of CBP/p300 and BET proteins attenuates TRPM3-mediated activation of AP-1. ( A ) Provirus containing the Coll.luc (collagenase promoter/luciferase) reporter gene. The AP-1 DNA binding site (known as 12- O -tetradecanoylphorbol-13-acetate (TPA)-responsive element (TRE)) is indicated. The provirus contains a deletion within the 5′LTR U3 region. Additionally, the provirus contains the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) and the HIV flap element. ( B , C ) T-REx-TRPM3 cells were infected with a Coll.luc-containing recombinant lentivirus. Cells were incubated in a serum-reduced medium for 24 h that was supplemented with tetracycline (1 μg/mL) to induce TRPM3 expression. Preincubation of the cells was performed for 3 h with either the histone acetyltransferase inhibitor A485 (3 μM) ( B ), or the BET inhibitor JQ1 (0.5 μM) ( C ). T-REx-TRPM3 cells were stimulated for 24 h with pregnenolone sulfate (PregS, 20 μM) in the presence of the inhibitor. Cell extracts were prepared and analyzed for luciferase activities. Luciferase activity was normalized to the protein concentration. Data shown are mean +/− SD of four ( B ) or three ( C ) independent experiments performed in quadruplicate (*** p < 0.001).

Article Snippet: The BET bromodomain inhibitor JQ1 (PubChem ID 46907787),(6 S )-4-(4-Chlorophenyl)-2,3,9-trimethyl-6 H -thieno [3,2- f ][1,2,4]triazolo [4,3- a ][1,4]diazepine-6-acetic acid 1,1-dimethylethyl ester) was purchased from Adooq Bioscience (Irvine, CA, USA, #A12729), dissolved in DMSO and used at a final concentration of 0.5 μM.

Techniques: Inhibition, Activation Assay, Luciferase, Binding Assay, Virus, Infection, Recombinant, Incubation, Expressing, Activity Assay, Protein Concentration

Journal: Pharmaceuticals

Article Title: TRPM3-Induced Gene Transcription Is under Epigenetic Control

doi: 10.3390/ph15070846

Figure Lengend Snippet: Pharmacological inhibition of CBP/p300 and BET proteins attenuates TRPM3-induced upregulation of the transcriptional activation potential of c-Fos. ( A ) The modular structure of the GAL4-c-Fos fusion protein. ( B ) Provirus that encodes the GAL4-responsive reporter gene UAS 5 Sp1 2 .luc. ( C , D ) T-REx-TRPM3 cells were infected with a UAS 5 Sp1 2 .luc containing lentivirus. T-REx-TRPM3 cells were infected with a second lentivirus that encoded for the GAL4-c-Fos protein. The cells were maintained in the serum-reduced medium in the presence of tetracycline for 24 h. T-REx-TRPM3 cells were stimulated with pregnenolone sulfate (PregS, 20 μM) for 24 h in the presence or absence of A485 (3 μM) ( C ), or JQ1 (0.5 μM) ( D ). Cells were harvested and analyzed as described in the legend to (( C ), n = 6; ( D ), n = 3; *** p < 0.001).

Article Snippet: The BET bromodomain inhibitor JQ1 (PubChem ID 46907787),(6 S )-4-(4-Chlorophenyl)-2,3,9-trimethyl-6 H -thieno [3,2- f ][1,2,4]triazolo [4,3- a ][1,4]diazepine-6-acetic acid 1,1-dimethylethyl ester) was purchased from Adooq Bioscience (Irvine, CA, USA, #A12729), dissolved in DMSO and used at a final concentration of 0.5 μM.

Techniques: Inhibition, Activation Assay, Infection

Journal: Pharmaceuticals

Article Title: TRPM3-Induced Gene Transcription Is under Epigenetic Control

doi: 10.3390/ph15070846

Figure Lengend Snippet: CBP/p300 and BET inhibitors reduce the transcriptional activation potential of Elk-1 in stimulated T-REx-TRPM3 cells. ( A ) Domain structure of GAL4-Elk-1 ( B , C ) T-REx-TRPM3 cells were infected with a lentivirus containing the UAS 5 Sp1 2 .luc reporter gene. Cells were infected with a second lentivirus that encoded for the GAL4-Elk-1 fusion protein. T-REx-TRPM3 cells were maintained in serum-reduced and tetracyclin-supplemented medium for 24 h. Stimulation was performed with pregnenolone sulfate (PregS, 20 μM) for 24 h. Addition of either A485 (3 μM) ( B ) or JQ1 (0.5 μM) ( C ) is indicated. Cells were harvested and analyzed as described in the legend to ( n = 3; *** p < 0.001).

Article Snippet: The BET bromodomain inhibitor JQ1 (PubChem ID 46907787),(6 S )-4-(4-Chlorophenyl)-2,3,9-trimethyl-6 H -thieno [3,2- f ][1,2,4]triazolo [4,3- a ][1,4]diazepine-6-acetic acid 1,1-dimethylethyl ester) was purchased from Adooq Bioscience (Irvine, CA, USA, #A12729), dissolved in DMSO and used at a final concentration of 0.5 μM.

Techniques: Activation Assay, Infection

Journal: Pharmaceuticals

Article Title: TRPM3-Induced Gene Transcription Is under Epigenetic Control

doi: 10.3390/ph15070846

Figure Lengend Snippet: Pharmacological inhibition of CBP/p300 and BET bromodomain proteins attenuates TRPM3-induced activation of CREB. ( A ) Provirus containing the c-FosCRE 4 .luc gene. The sequence of the CRE is shown. ( B ) Domain structures of CREB, CREB2 and C2/CREB. KID, kinase inducible domain; Q1, Q2, glutamine-rich activation domains of CREB. ( C ) T-REx-TRPM3 cells were infected with a lentivirus encoding either C2/CREB or β-galactosidase (mock). Additionally, lentiviral gene transfer was used to integrate the c-FosCRE 4 .luc reporter gene into the chromatin. Infected T-REx-TRPM3 cells were maintained for 3 days. Cell extracts were prepared, luciferase activities were determined and protein concentrations were measured. The protein concentration were used to normalize the luciferase activities ( n = 4; *** p < 0.001). ( D , E ) T-REx-TRPM3 cells containing a chromatin-embedded c-FosCRE 4 .luc reporter gene were maintained in serum-reduced and tetracycline-supplemented medium for 24 h. Stimulation was performed with pregnenolone sulfate for 24 h. Additon of either A485 (3 μM) ( D ) or JQ1 (0.5 μM) ( E ) to the culture medium is indicated. T-REx-TRPM3 cells were harvested and analyzed as described in the legend to ( n = 3; ** p < 0.01; *** p < 0.001).

Article Snippet: The BET bromodomain inhibitor JQ1 (PubChem ID 46907787),(6 S )-4-(4-Chlorophenyl)-2,3,9-trimethyl-6 H -thieno [3,2- f ][1,2,4]triazolo [4,3- a ][1,4]diazepine-6-acetic acid 1,1-dimethylethyl ester) was purchased from Adooq Bioscience (Irvine, CA, USA, #A12729), dissolved in DMSO and used at a final concentration of 0.5 μM.

Techniques: Inhibition, Activation Assay, Sequencing, Infection, Luciferase, Protein Concentration

Journal: Pharmaceuticals

Article Title: TRPM3-Induced Gene Transcription Is under Epigenetic Control

doi: 10.3390/ph15070846

Figure Lengend Snippet: Regulation of epigenetic gene transcription by CBP/p300 and BET bromodomain proteins.

Article Snippet: The BET bromodomain inhibitor JQ1 (PubChem ID 46907787),(6 S )-4-(4-Chlorophenyl)-2,3,9-trimethyl-6 H -thieno [3,2- f ][1,2,4]triazolo [4,3- a ][1,4]diazepine-6-acetic acid 1,1-dimethylethyl ester) was purchased from Adooq Bioscience (Irvine, CA, USA, #A12729), dissolved in DMSO and used at a final concentration of 0.5 μM.

Techniques:

Journal: Oncotarget

Article Title: Generation and characteristics of a novel “double-hit” high grade B-cell lymphoma cell line DH-My6 with MYC / IGH and BCL6 / IGH gene arrangements and potential molecular targeted therapies

doi: 10.18632/oncotarget.26060

Figure Lengend Snippet: (A) Cell growth assay following treatment with the indicated doses of JQ1. (B) Cell cycle analysis conducted at 48 h after treatment with JQ1 (250 nM). (C) Apoptosis assay conducted at 48 h after treatment with the indicated doses of JQ1. (D) Cell growth assay following treatment with the indicated doses of FX1. (E) Cell cycle analysis conducted at 48 h after treatment with FX1 (25 μM). (F) Apoptosis assay conducted at 48 h after treatment with the indicated doses of FX1. (G) Cell growth assay following treatment with the various doses of JQ1, FX1, and their combinations for 72 h. The numbers of viable cells are normalized as a percentage of the viable cell numbers of DMSO-treated controls. (H) Apoptosis assay conducted at 48 h after a single treatment with JQ1 (500 nM), FX1 (50 μM), and their combination at each concentration that gave a limited effect on apoptosis when treated with a single agent. Data are shown as the mean ± SEM of three independent experiments. Significant expression differences are shown as * P < 0.05; ** P <0.01.

Article Snippet: The BET domain inhibitor JQ1 was obtained from Merck KGaA, the HDAC inhibitor vorinostat was from Cayman Chemical, and the PLK1 inhibitor volasertib was from ChemScene (Monmouth Junction, NJ, USA).

Techniques: Growth Assay, Cell Cycle Assay, Apoptosis Assay, Concentration Assay, Expressing

Journal: Oncotarget

Article Title: Generation and characteristics of a novel “double-hit” high grade B-cell lymphoma cell line DH-My6 with MYC / IGH and BCL6 / IGH gene arrangements and potential molecular targeted therapies

doi: 10.18632/oncotarget.26060

Figure Lengend Snippet: Estimated IC 50 values in the lymphoma cell lines treated with inhibitors for 72 h

Article Snippet: The BET domain inhibitor JQ1 was obtained from Merck KGaA, the HDAC inhibitor vorinostat was from Cayman Chemical, and the PLK1 inhibitor volasertib was from ChemScene (Monmouth Junction, NJ, USA).

Techniques:

Journal: Oncotarget

Article Title: Generation and characteristics of a novel “double-hit” high grade B-cell lymphoma cell line DH-My6 with MYC / IGH and BCL6 / IGH gene arrangements and potential molecular targeted therapies

doi: 10.18632/oncotarget.26060

Figure Lengend Snippet: (A) Cell growth assay following treatment with the indicated doses of volasertib. (B) Cell cycle analysis conducted at 48 h after treatment with volasertib (5 nM). (C) Apoptosis assay conducted at 48 h after treatment with the indicated doses of volasertib. (D) Cell growth assay following transfection with PLK1 or control siRNAs. (E) Cell cycle analysis conducted at 48 h after siRNA transfection. (F) Apoptosis assay conducted at 48 h after siRNA transfection. (G) Cell growth assay following treatment with the various doses of volasertib, JQ1, and their combinations for 72 h. (H) Apoptosis assay conducted at 48 h after a single treatment with volasertib (5 nM), JQ1 (500 nM), and their combination at doses that did not induce significant apoptosis individually. (I) Cell growth assay following treatment with the various doses of volasertib, FX1, and their combinations for 72 h. (J) Apoptosis assay conducted at 48 h after a single treatment with volasertib (5 nM), FX1 (50 μM), and their combination at doses that did not induce significant apoptosis individually. Data are shown as the mean ± SEM of three independent experiments. Significant expression differences are shown as * P < 0.05; ** P <0.01.

Article Snippet: The BET domain inhibitor JQ1 was obtained from Merck KGaA, the HDAC inhibitor vorinostat was from Cayman Chemical, and the PLK1 inhibitor volasertib was from ChemScene (Monmouth Junction, NJ, USA).

Techniques: Growth Assay, Cell Cycle Assay, Apoptosis Assay, Transfection, Control, Expressing

Journal: Oncotarget

Article Title: Generation and characteristics of a novel “double-hit” high grade B-cell lymphoma cell line DH-My6 with MYC / IGH and BCL6 / IGH gene arrangements and potential molecular targeted therapies

doi: 10.18632/oncotarget.26060

Figure Lengend Snippet: (A) Cell growth assay following treatment with the indicated doses of vorinostat. (B) Cell cycle analysis conducted at 48 h after treatment with vorinostat (1 μM). (C) Apoptosis assay conducted at 48 h after treatment with the indicated doses of vorinostat. (D) Cell growth assay following treatment with the various doses of vorinostat, JQ1, and their combinations for 72 h. (E) Apoptosis assay conducted at 48 h after a single treatment with vorinostat (500 nM), JQ1 (500 nM), and their combination at doses that did not induce significant apoptosis individually. (F) Cell growth assay following treatment with the various doses of vorinostat, FX1, and their combinations for 72 h. (G) Apoptosis assay conducted at 48 h after single treatment with vorinostat (500 nM), FX1 (25 μM), and their combination at doses that did not induce significant apoptosis individually. (H) Cell growth assay following treatment with the various doses of vorinostat, volasertib, and their combinations for 72 h. (I) Apoptosis assay conducted at 48 h after a single treatment with vorinostat (500 nM), volasertib (2.5 nM), and their combination at doses that did not induce significant apoptosis individually. Data are shown as the mean ± SEM of three independent experiments. Significant expression differences are shown as * P < 0.05; ** P <0.01.

Article Snippet: The BET domain inhibitor JQ1 was obtained from Merck KGaA, the HDAC inhibitor vorinostat was from Cayman Chemical, and the PLK1 inhibitor volasertib was from ChemScene (Monmouth Junction, NJ, USA).

Techniques: Growth Assay, Cell Cycle Assay, Apoptosis Assay, Expressing