Journal: Cell Reports

Article Title: BET Inhibition Induces HEXIM1- and RAD51-Dependent Conflicts between Transcription and Replication

doi: 10.1016/j.celrep.2018.10.079

Figure Lengend Snippet: BET Inhibition Induces Replication-Transcription Conflicts (A) DNA fiber labeling in U2OS cells treated with JQ1. (B) Replication fork speeds after JQ1 treatment (n = 3–6). (C) EU labeling after JQ1 treatment. (D) Representative images of click-stained EU labeled cells ± 8 hr JQ1. (E) Nuclear EU intensities after JQ1 treatment (n = 3–5). (F) RNA was extracted after 8 hr JQ1 treatment and yield normalized to cell number and DMSO (n = 7). (G) Fold change in the normalized expression levels of indicated transcripts ± JQ1 as indicated (n = 4). (H) Cells were treated with transcription inhibitors before and during EU or DNA fiber labeling. AM, α-amanitin; TRIP, triptolide. (I) Nuclear EU intensities in cells treated with transcription inhibitors and JQ1 (n = 4). (J) Replication fork speeds after 1 hr JQ1 ± transcription inhibitors (n = 3 or 4). (K) JQ1 effect on nascent RNA synthesis and replication fork speeds in a panel of human cell lines. Data are represented as mean ± SEM. Scale bars, 10 μm. See also and .

Article Snippet: Triptolide , Tocris , 3253/1.

Techniques: Inhibition, Labeling, Staining, Expressing

Journal: Cell Reports

Article Title: BET Inhibition Induces HEXIM1- and RAD51-Dependent Conflicts between Transcription and Replication

doi: 10.1016/j.celrep.2018.10.079

Figure Lengend Snippet:

Article Snippet: Triptolide , Tocris , 3253/1.

Techniques: Recombinant, Imaging, Reverse Transcription, Gene Expression, Sequencing, Negative Control, Software

Journal: bioRxiv

Article Title: Human RNase H2 upregulation counteracts oncogene- and chemotherapy-induced replication stress

doi: 10.1101/2024.12.16.628316

Figure Lengend Snippet: (A) RNase H2 complex subunits and functions. (B) Protein levels of HRAS, RNASEH2B (RH2B), RNASEH2C (RH2C) and Vinculin (loading control) in BJ-hTERT HRAS V12ERTAM cells +/-72 h HRAS G12V induction with 4-hydroxytamoxifen (4-OHT). (C) Relative protein levels of RH2B and RH2C after 24, 72 or 96 h HRAS G12V induction normalised to ethanol (con) for each protein. N=5 (24 and 96 h), N=13 (RH2B 72 h), N=6 (RH2C 72 h). (D) RT-qPCR analysis of RNASEH2A, RNASEH2B , and RNASEH2C expression after HRAS G12V induction. Asterisks compare to con. N = 4 (24 h), N = 5 (48 and 72 h). (E) Substrates used for the RNase H2 activity assay with forward strand covalently coupled to 3’-fluorescein (green) and reverse strand linked to 5’-DABCYL quencher (orange). (F) Representative time course of DRD:DNA substrate conversion during incubation with whole cell extract +/- 72 h HRAS G12V induction (control). N = 2 measured on the same plate. (G) Relative RNase H2 activities in cell extracts +/- 72 h HRAS G12V induction. N = 4. (H) Protein levels of CYCLIN E, RH2B, RH2C and Tubulin (loading control) in U2OS-CYCLIN E cells +/-CYCLIN E induction with tetracycline removal (-tet) for the times indicated. (I) Protein levels of RH2B and Tubulin (loading control) in BJ-hTERT HRAS V12ERTAM cells without HRAS G12V induction after 4 h treatment with 200 μM hydroxyurea (HU), 25 nM gemcitabine (GEM), 10 μM camptothecin (CPT) or DMSO (con). Means and SEM (bars) of independent experiments are shown. Asterisks indicate p-values (ANOVA or mixed-effects analysis, * p < 0.05, ** p < 0.01).

Article Snippet: Gemcitabine and triptolide were from Tocris Bioscience.

Techniques: Control, Quantitative RT-PCR, Expressing, Activity Assay, Incubation

Journal: Archives of Toxicology

Article Title: How to avoid misinterpretation of dual reporter gene assay data affected by cell damage

doi: 10.1007/s00204-022-03323-0

Figure Lengend Snippet: Relative PXR reporter activity and firefly or Renilla luminescence with a PXR inhibitor exhibiting low cytotoxicity. Idealized model (upper panels): a ① Proliferation is neither affected by the activator alone (e.g., rifampicin) nor by the proposed PXR inhibitor being added to the activator; ② PXR ligand-mediated enhancement of relative PXR activity (e.g. threefold compared to untreated control, dashed line) is concentration-dependently abolished by proposed PXR inhibitor being added to the activator. Eventually, relative PXR activity returns to the baseline level. b ① Strong PXR activator-mediated increase of the firefly luminescence (white circle), but unchanged Renilla signal (white square); ② Addition of an inhibitor leads to a sigmoidal decrease of the firefly luminescence; ③ Renilla luminescence remains constant. Experimental data (lower panels): c Impact of triptolide on cell proliferation and relative PXR activity when added to 5 µM rifampicin (24 h drug exposure). d Firefly and Renilla luminescence normalized to untreated control. Data shown is the mean ± SEM of three independent biological replicates with n = 4 (reporter data) or n = 8 (proliferation data) replicates for each concentration/replicate. Impact of drug treatments on firefly or Renilla values was evaluated by ANOVA with non-parametric Kruskal–Wallis test and Dunn’s test compared to untreated control. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Triptolide was purchased from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Activity Assay, Control, Concentration Assay

Journal: PLoS Genetics

Article Title: Drosophila TDP-43 RNA-Binding Protein Facilitates Association of Sister Chromatid Cohesion Proteins with Genes, Enhancers and Polycomb Response Elements

doi: 10.1371/journal.pgen.1006331

Figure Lengend Snippet: The boxplots show the average ChIP-seq enrichment of Ser5P Pol II, Nipped-B, TBPH and Lark at active promoters, extragenic enhancers, and PREs in control cells, and cells treated with 10 μM triptolide for 1, 2, and 4 hours. The genome browser tracks at the right show the log2 ChIP-seq enrichment for the same proteins at the string gene and enhancers over the same time course of triptolide treatment.

Article Snippet: The Ser5P Pol II rabbit monoclonal antibody was purchased from Cell Signaling Technology (#13523) and validated by western blots of cell extracts with and without triptolide treatments.

Techniques: ChIP-sequencing, Control

Journal: PLoS Genetics

Article Title: Drosophila TDP-43 RNA-Binding Protein Facilitates Association of Sister Chromatid Cohesion Proteins with Genes, Enhancers and Polycomb Response Elements

doi: 10.1371/journal.pgen.1006331

Figure Lengend Snippet: The log2 ChIP-seq enrichment for Ser5P Pol II, Nipped-B, TBPH and Lark at all individual active promoters, enhancers and PREs in control untreated (Mock) cells is plotted against the enrichment after treatment of cells with 10 μM triptolide for 1, 2 and 4 hours.

Article Snippet: The Ser5P Pol II rabbit monoclonal antibody was purchased from Cell Signaling Technology (#13523) and validated by western blots of cell extracts with and without triptolide treatments.

Techniques: ChIP-sequencing, Control

Journal: Acta Pharmaceutica Sinica. B

Article Title: Establishment of interpretable cytotoxicity prediction models using machine learning analysis of transcriptome features

doi: 10.1016/j.apsb.2025.02.009

Figure Lengend Snippet: CTS genes facilitating the MOA study of the compounds with NTI. (A) The pipeline for identifying CTS genes in the RF models. (B, C) The MOAs for cyclosporine A (B) and triptolide (C) were predicted using DEGs of the compound and the DEGs with removal of CTS genes. The two sets of top 10 MOAs identified by using the compound DEGs (in lavender gray) and the DEGs associated with CTS gene removal (in apricot) are shown in the Venn diagram. The target-related terms are shown in red and marked with asterisks (∗∗∗). The ranking for each MOA term is indicated in parentheses. The MOA enrichment was analyzed using the Query platform ( https://clue.io/query ). BCL, B cell lymphoma; CDK, cyclin-dependent kinase; EGFR, epidermal growth factor receptor; FGFR, fibroblast growth factor receptor; FLT3, FMS-like tyrosine kinase 3; HDAC, histone deacetylase; HMGCR, 3-hydroxy-3-methylglutaryl-coenzyme A reductase; IGF-1, insulin-like growth factor 1; MEK, mitogen-activated extracellular signal-regulated kinase; PARP, poly(ADP-ribose) polymerase; PKC, protein kinase C.

Article Snippet: L1000, L3400, L6000, and L4000), and the powders of the compounds, cyclosporine A (Cat. No. T0945; purity = 98%) and triptolide (Cat. No. T2179; purity = 98%), were purchased from TargetMol (Boston, MA, USA).

Techniques: Histone Deacetylase Assay

Journal: Cell Death & Disease

Article Title: Triptolide induces apoptosis in human leukemia cells through caspase-3-mediated ROCK1 activation and MLC phosphorylation

doi: 10.1038/cddis.2013.469

Figure Lengend Snippet: Triptolide induced apoptosis and mitochondrial injury in multiple leukemia cell lines. ( a ) The chemical structure of triptolide, C 20 H 24 O 6 , molecular weight: 360.4. ( b and c ) U937 cells were treated with various triptolide (TPL) concentrations for 24 h or with 40 nM triptolide for different lengths. The percentage of apoptotic cells was determined by FACS analysis using Annexin V/PI staining. Mitochondrial membrane potentials (ΔΨm) were detected by rhodamine-123 staining and flow cytometry. Values represent the mean±S.D. for five separate experiments. Total protein lysates, nuclear extracts, and cytosolic fractions were analyzed by immunoblotting using the indicated antibodies. ( d ) After triptolide treatment, cells were collected and stained with anti-AIF (green) and 4′,6-diamidino-2-phenylindole (DAPI; blue) to identify cellular nuclei. Fluorescence was visualized by a laser confocal scanning microscope. Scale bar represents 10 μ m. These data are representative of three independent experiments. ( e and f ) U937, Jurkat, and HL-60 cells were treated with or without 40 nM triptolide for 24 h, after which apoptosis was determined by FACS analysis using Annexin V/PI staining. Total protein lysates, nuclear extracts, and cytosolic fractions were analyzed by immunoblotting using the indicated antibodies. CF, cleavage fragment; C, cytosolic fractions; N, nuclear extracts

Article Snippet: Triptolide was purchased from Alexis Biochemicals (San Diego, CA, USA); z-Asp-Glu-Vad-Asp(Ome)-fluoromethylketone (z-DEVD-fmk) and z-Val-Ala-Asp(Ome)-fluoromethylketone (z-VAD-fmk) from Enzyme Systems Products (Livermore, CA, USA); exoenzyme C3 and 1-(5-iodonapthalene-1-sulfonyl) homopiperazine hydrochloride (ML-7) from Calbiochem (La Jolla, CA, USA); and Y-27632 from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Molecular Weight, Staining, Flow Cytometry, Western Blot, Fluorescence, Microscopy

Journal: Cell Death & Disease

Article Title: Triptolide induces apoptosis in human leukemia cells through caspase-3-mediated ROCK1 activation and MLC phosphorylation

doi: 10.1038/cddis.2013.469

Figure Lengend Snippet: Triptolide induced apoptosis in primary human leukemia blasts but not in normal CD34 + cells. ( a ) Primary leukemia blasts from 44 patients were treated with 40 nM triptolide for 24 or 36 h, after which apoptosis was determined by FACS analysis using Annexin V/PI staining. Upper panel: representative FACS images. Lower panel: numbers indicate the percentage of apoptotic cells; each symbol represents results from individual patients. ( b ) Total protein lysates and cytosolic fractions of the samples from four AML patients were then obtained and analyzed by immunoblotting using the indicated antibodies. ( c ) Normal CD34 + cells were isolated from 6 normal bone marrow samples and exposed to 40 nM triptolide for 24 or 36 h. The percentage of apoptotic cells was determined by FACS analysis using Annexin V/PI staining. ( d ) Two normal donors were treated with 40 nM triptolide for 24 or 36 h. Total protein lysates and cytosolic fractions of these samples were then obtained and analyzed by immunoblotting using the indicated antibodies

Article Snippet: Triptolide was purchased from Alexis Biochemicals (San Diego, CA, USA); z-Asp-Glu-Vad-Asp(Ome)-fluoromethylketone (z-DEVD-fmk) and z-Val-Ala-Asp(Ome)-fluoromethylketone (z-VAD-fmk) from Enzyme Systems Products (Livermore, CA, USA); exoenzyme C3 and 1-(5-iodonapthalene-1-sulfonyl) homopiperazine hydrochloride (ML-7) from Calbiochem (La Jolla, CA, USA); and Y-27632 from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Staining, Western Blot, Isolation

Journal: Cell Death & Disease

Article Title: Triptolide induces apoptosis in human leukemia cells through caspase-3-mediated ROCK1 activation and MLC phosphorylation

doi: 10.1038/cddis.2013.469

Figure Lengend Snippet: Triptolide induced Bax translocation in multiple leukemia cell lines. ( a ) U937 cells were treated with various triptolide concentrations for 24 h or with 40 nM triptolide for different lengths, after which cytosolic and mitochondrial fractions were isolated and subjected to immunoblot analysis by using an anti-Bax antibody. ( b ) U937 cells were treated with or without 40 nM triptolide for 24 h. Cells were collected and stained with anti-Bax (green) and MitoTracker (red) to identify mitochondria. Fluorescence was visualized by a laser confocal scanning microscope. Scale bar represents 10 μ m. These data are representative of three independent experiments. ( c ) U937, Jurkat, and HL-60 cells were treated with or without 40 nM triptolide for 24 h, after which cytosolic and mitochondrial fractions were isolated and subjected to immunoblot analysis by using an anti-Bax antibody

Article Snippet: Triptolide was purchased from Alexis Biochemicals (San Diego, CA, USA); z-Asp-Glu-Vad-Asp(Ome)-fluoromethylketone (z-DEVD-fmk) and z-Val-Ala-Asp(Ome)-fluoromethylketone (z-VAD-fmk) from Enzyme Systems Products (Livermore, CA, USA); exoenzyme C3 and 1-(5-iodonapthalene-1-sulfonyl) homopiperazine hydrochloride (ML-7) from Calbiochem (La Jolla, CA, USA); and Y-27632 from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Translocation Assay, Isolation, Western Blot, Staining, Fluorescence, Microscopy

Journal: Cell Death & Disease

Article Title: Triptolide induces apoptosis in human leukemia cells through caspase-3-mediated ROCK1 activation and MLC phosphorylation

doi: 10.1038/cddis.2013.469

Figure Lengend Snippet: Triptolide induced ROCK1 activation and MLC and MYPT phosphorylation. ( a ) U937 cells were treated with various triptolide concentrations for 24 h or with 40 nM triptolide for different lengths. Total protein lysates were analyzed by immunoblotting using the indicated antibodies. RhoA-GTP was evaluated using a Rhotekin RBD-GST pull-down. ( b ) U937, Jurkat, and HL-60 cells were treated with or without 40 nM triptolide for 24 h. Total protein lysates were analyzed by immunoblotting using the indicated antibodies. Samples from four AML patients ( c ) and two normal donors ( d ) were treated with 40 nM triptolide for 24 or 36 h. The total protein lysates of these samples were obtained and analyzed by immunoblotting using the indicated antibodies

Article Snippet: Triptolide was purchased from Alexis Biochemicals (San Diego, CA, USA); z-Asp-Glu-Vad-Asp(Ome)-fluoromethylketone (z-DEVD-fmk) and z-Val-Ala-Asp(Ome)-fluoromethylketone (z-VAD-fmk) from Enzyme Systems Products (Livermore, CA, USA); exoenzyme C3 and 1-(5-iodonapthalene-1-sulfonyl) homopiperazine hydrochloride (ML-7) from Calbiochem (La Jolla, CA, USA); and Y-27632 from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Activation Assay, Western Blot

Journal: Cell Death & Disease

Article Title: Triptolide induces apoptosis in human leukemia cells through caspase-3-mediated ROCK1 activation and MLC phosphorylation

doi: 10.1038/cddis.2013.469

Figure Lengend Snippet: Caspase-3, rather than RhoA, activated ROCK1 during triptolide-induced apoptosis. Caspase inhibitors (z-VAD-fmk or z-DEVD-fmk) and a Rho inhibitor (C3 exoenzyme) were used to explore the mechanism behind ROCK1 activation. The percentage of apoptotic cells was determined by FACS analysis using Annexin V/PI staining. Error bars represent the mean±S.D. ( n =5). Total protein lysates were analyzed by immunoblotting using the indicated antibodies. Results are representative of three independent experiments. ( a–c ) U937 cells and blasts from one AML patient were pretreated with 20 μ M z-VAD-fmk or z-DEVD-fmk for 2 h followed by treatment with or without 40 nM triptolide for 24 h. ( d – f ) U937 cells and blasts from one AML patient were transfected with 5 μ g C3 (premixed with FuGENE 6 Reagent in RPMI-1640 medium). After 5 h of incubation, cells were treated with or without 40 nM triptolide for 24 h

Article Snippet: Triptolide was purchased from Alexis Biochemicals (San Diego, CA, USA); z-Asp-Glu-Vad-Asp(Ome)-fluoromethylketone (z-DEVD-fmk) and z-Val-Ala-Asp(Ome)-fluoromethylketone (z-VAD-fmk) from Enzyme Systems Products (Livermore, CA, USA); exoenzyme C3 and 1-(5-iodonapthalene-1-sulfonyl) homopiperazine hydrochloride (ML-7) from Calbiochem (La Jolla, CA, USA); and Y-27632 from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Activation Assay, Staining, Western Blot, Transfection, Incubation

Journal: Cell Death & Disease

Article Title: Triptolide induces apoptosis in human leukemia cells through caspase-3-mediated ROCK1 activation and MLC phosphorylation

doi: 10.1038/cddis.2013.469

Figure Lengend Snippet: Inhibiting MLC phosphorylation significantly decreased triptolide-induced apoptosis. U937 cells and blasts from one AML patient were pretreated with 20 μ M ML-7, a MLC kinase inhibitor, for 2 h followed by treatment with or without 40 nM triptolide for 24 h. ( a ) The percentage of apoptotic cells was determined by FACS analysis using Annexin V/PI staining. Error bars represent the mean±S.D. ( n =5). ( b and c ) Total protein lysates and cytosolic fractions were analyzed by immunoblotting using the indicated antibodies. Results were representative of three independent experiments

Article Snippet: Triptolide was purchased from Alexis Biochemicals (San Diego, CA, USA); z-Asp-Glu-Vad-Asp(Ome)-fluoromethylketone (z-DEVD-fmk) and z-Val-Ala-Asp(Ome)-fluoromethylketone (z-VAD-fmk) from Enzyme Systems Products (Livermore, CA, USA); exoenzyme C3 and 1-(5-iodonapthalene-1-sulfonyl) homopiperazine hydrochloride (ML-7) from Calbiochem (La Jolla, CA, USA); and Y-27632 from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Staining, Western Blot

Journal: Cell Death & Disease

Article Title: Triptolide induces apoptosis in human leukemia cells through caspase-3-mediated ROCK1 activation and MLC phosphorylation

doi: 10.1038/cddis.2013.469

Figure Lengend Snippet: Inhibiting ROCK1 activation attenuated triptolide-induced apoptosis. ( a ) U937 cells and blasts from one AML patient were pretreated with 20 μ M Y-27632, a ROCK1 inhibitor, for 2 h followed by treatment with 40 nM triptolide for 24 h. The percentage of apoptotic cells was determined by FACS analysis using Annexin V/PI staining. Error bars represent the mean±S.D. ( n =5). ( b and c ) Total protein lysates and cytosolic fractions were analyzed by immunoblotting using the indicated antibodies. Results are representative of three independent experiments. ( d ) U937 cells were stably transfected with lentivirus containing ROCK1-specific siRNA (si-ROCK1) or a scrambled control siRNA. Cells were treated with or without 40 nM triptolide for 24 h, and apoptosis was measured by FACS analysis using Annexin V/PI staining. Error bars represent the mean±S.D. ( n =5). ( e and f ) Total protein lysates and cytosolic fractions were analyzed by immunoblotting using the indicated antibodies. Results are representative of three independent experiments

Article Snippet: Triptolide was purchased from Alexis Biochemicals (San Diego, CA, USA); z-Asp-Glu-Vad-Asp(Ome)-fluoromethylketone (z-DEVD-fmk) and z-Val-Ala-Asp(Ome)-fluoromethylketone (z-VAD-fmk) from Enzyme Systems Products (Livermore, CA, USA); exoenzyme C3 and 1-(5-iodonapthalene-1-sulfonyl) homopiperazine hydrochloride (ML-7) from Calbiochem (La Jolla, CA, USA); and Y-27632 from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Activation Assay, Staining, Western Blot, Stable Transfection, Transfection

Journal: Cell Death & Disease

Article Title: Triptolide induces apoptosis in human leukemia cells through caspase-3-mediated ROCK1 activation and MLC phosphorylation

doi: 10.1038/cddis.2013.469

Figure Lengend Snippet: Triptolide inhibited the growth of U937 xenografts. A total of 24 nude mice were inoculated with U937 cells and randomly divided into two groups (12 mice/group) that were treated with either vehicle or triptolide. ( a ) Tumor volumes were measured at the indicated intervals (upper panel) and images are shown for five representative tumors from each group after 40 days of treatment (lower panel). Data are shown as the mean±S.D. * P <0.05; ** P <0.01; *** P <0.001. ( b ) Changes in body weight in the mice during 40 days of triptolide treatment. ( c ) Tumors were fixed and stained with H&E to examine tumor cell morphology. TUNEL assays were used to determine the apoptotic effects of triptolide. Immunohistochemistry was used to determine the levels of cleaved caspase-3 and phospho-MLC. Scale bar represents 50 μ m. ( d ) After treatment, tumors from the vehicle and triptolide groups were lysed and subjected to immunoblotting with anti-ROCK1. Results are representative of three independent experiments. ( e ) An illustration of the molecular mechanism of triptolide-induced apoptosis. Exposure to triptolide results in activation of ROCK1 and MLC and MYPT phosphorylation, leading in turn to Bax translocation, culminating in cytochrome c release (Cyto c ), caspases activation, and apoptosis. Activated caspase-3 in turn causes activation of ROCK, thus creating a positive feedback loop

Article Snippet: Triptolide was purchased from Alexis Biochemicals (San Diego, CA, USA); z-Asp-Glu-Vad-Asp(Ome)-fluoromethylketone (z-DEVD-fmk) and z-Val-Ala-Asp(Ome)-fluoromethylketone (z-VAD-fmk) from Enzyme Systems Products (Livermore, CA, USA); exoenzyme C3 and 1-(5-iodonapthalene-1-sulfonyl) homopiperazine hydrochloride (ML-7) from Calbiochem (La Jolla, CA, USA); and Y-27632 from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Staining, TUNEL Assay, Immunohistochemistry, Western Blot, Activation Assay, Translocation Assay