Journal: Natural Sciences

Article Title: Progenitor‐derived ribosomal RNA supports protein synthesis in Drosophila neurons

doi: 10.1002/ntls.20210032

Figure Lengend Snippet: FIGURE 1 Cell type-specific biosynthetic RNA tagging reveals limited rRNA synthesis in neurons. (a) Fluorescent RNA signal (green) in a single L3-stage brain lobe following 24 h of EC-tagging targeted to neural progenitors (insc > CD:UPRT) or neurons (nSyb > CD:UPRT). Scale bar is 10 µm. A single confocal slice encompassing the entire brain lobe is shown. Images are representative of replicate experiments in which a minimum of eight brain lobes were analyzed per condition. (b) Relative precursor rRNA (pre-rRNA) and Syt1 transcript abundance following 24 h of EC-tagging (starting at 48–49 h after larval hatching (ALH) and ending at 72–73 hours ALH) in neurons (nSyb-tag) or neural progenitors (insc-tag), as measured by RT-qPCR. Fold-change in relative abundance (nSyb-tag / insc-tag), after normalization to a RNA polymerase II subunit, is shown. Data are the mean and standard deviation from duplicate RT-qPCR reactions performed on biological replicate EC-tagging experiments. *p-value < 0.05, ***p-value < 0.001, Student’s t-test. (c) The fluorescent signal is predominately ribosomal RNA. Brains dissected from middle L3-stage larvae were soaked in EU alone (left panel), EU plus the mRNA synthesis inhibitor triptolide (middle panel), or EU plus the mRNA and rRNA synthesis inhibitor actinomycin D (right panel). An equivalent region of central brain (excluding the optic lobe) is shown in each confocal stack. Scale bar is 10 µm. Images are representative of replicate experiments in which a minimum of eight brain lobes were analyzed per condition. (d) The EU-RNA signal localizes to the nucleolus of neuroblasts. EU-RNA signal alone (left panel), overlay of EU-RNA and the nucleolus marker Udd (middle panel), overlay of EU-RNA and the proliferating cell marker PCNA (right panel). All panels are from the same confocal stack. The large PCNA positive cells are neuroblasts. A confocal projection from the dorsal region of a single brain lobe is shown. Scale bar is 10 µm. The image is representative of results obtained from replicate experiments in which eight brain lobes were analyzed

Article Snippet: Triptolide (ThermoFisher) was used at a final concentration of 100 μM, 10-fold higher than the concentration known to inhibit RNA polymerase II in Drosophila tissue culture cells.27 Actinomycin D (Millipore Sigma) was used at a final concentration of 700 μM, a concentration that is expected to affect RNA polymerase I and II.21 For all imaging experiments, brains were fixed in 4% paraformaldehyde prior to Alexa Fluor 488 addition using the ClickiT RNA Imaging Kit (ThermoFisher), as previously described, for ClickiT kit-based detection of DNA labeled with 5-ethynyl-2′-deoxyuridine in Drosophila larval brains.38 The Click-iT reaction was followed by antibody staining according to standard methods.39 Imaging was performed using a Zeiss LSM 880 confocal microscope.

Techniques: Quantitative RT-PCR, Standard Deviation, Marker

Journal: Chinese Medicine

Article Title: Triptolide targets PPP2CA/ITGA5 axis to suppress lactate-driven ovarian cancer progression

doi: 10.1186/s13020-025-01174-2

Figure Lengend Snippet: Exosomal transfer of ITGA5 promotes tumor cell preference for metastasis toward HPMCs. A Western blot analysis of ITGA5 expression in SKOV3 cells after 48 h treatment with conditioned medium from ITGA5 -overexpressing cells. B Wound healing assay of SKOV3 cells cultured in conditioned medium from ITGA5 -overexpressing (oeITGA5) cells or control medium. C Electron microscopy images of exosomes derived from ITGA5 -overexpressing cells, nanoparticle tracking analysis showing the size distribution of the exosomes. D Western blot analysis of exosomes isolated from control and ITGA5 -overexpressing cells, detecting exosome markers (CD81, CD9, TSG101) and ITGA5 expression. E Wound healing assay of SKOV3 cells treated with exosomes isolated from ITGA5 -overexpressing cells or control cells. F , G Migration assay of HPMCs treated with exosomes isolated from various cell lines ( F ) or co-cultured with cells with ITGA5 knockdown or ITGA5/ITGB1 double knockdown ( G ). H, I Wound healing assays were performed on SKOV3 and OVCAR8 cells treated with Triptolide at different concentrations for 24 h, as well as on SKOV3 and OVCAR8 cells overexpressing ITGA5 , following Triptolide treatment. ns not significant; *p < 0.05, **p < 0.01. Scale bar = 50 µm

Article Snippet: After several days of growth, when organoids were nearing maturation, different concentrations of Triptolide (Selleck, S3604) were added.

Techniques: Western Blot, Expressing, Wound Healing Assay, Cell Culture, Control, Electron Microscopy, Derivative Assay, Isolation, Migration, Knockdown

Journal: Chinese Medicine

Article Title: Triptolide targets PPP2CA/ITGA5 axis to suppress lactate-driven ovarian cancer progression

doi: 10.1186/s13020-025-01174-2

Figure Lengend Snippet: Triptolide exhibits efficacy against patient-derived OC organoids. A Western blot analysis of SKOV3 and OVCAR8 cells treated with Triptolide at different concentrations. B Immunofluorescence of Triptolide-treated OC organoids (#11) stained for DAPI (blue), ITGA5 (red), and ITGB1 (green). Scale bar = 100 µm. C Western blot analysis of Triptolide-treated organoids. D Lactate levels in Triptolide-treated OC cells. E – G Representative images and viability analysis of Triptolide-treated OC organoids. Calcein-AM staining (4 × and 10 × magnification) and luminescence assay assess viability. H – K Tumor growth assessment in PPP2CA -knockout xenografts. Representative images of tumors ( H ), tumor growth curves ( I ), tumor weight ( J ) and volume ( K ) at the experimental endpoint. ns not significant; *p < 0.05, **p < 0.01, ***p < 0.001. Scale bar = 50 µm

Article Snippet: After several days of growth, when organoids were nearing maturation, different concentrations of Triptolide (Selleck, S3604) were added.

Techniques: Derivative Assay, Western Blot, Immunofluorescence, Staining, Luminescence Assay, Knock-Out

Journal: Cells

Article Title: Post-Transcriptional Regulation of PARP7 Protein Stability Is Controlled by Androgen Signaling

doi: 10.3390/cells10020363

Figure Lengend Snippet: AR-dependent transcription is required for PARP7 stabilization. ( A ) PC3-Flag-AR/HA-PARP7 cells were treated with indicated androgens (black) and anti-androgens (red) for 6 h and analyzed by immunoblotting. DHT: dihydrotestosterone, ASD: androstenedione, DHEA: dehydroepiandrosterone, HO-Flutamide: hydroxyflutamide, ENZ: enzalutamide, CPA: cyproterone acetate. ( B ) PC3-Flag-AR/HA-PARP7 cells were treated with R1881 (2 nM) for 3 h, and then treated for an additional 3 h with R1881 in the presence or absence of ENZ (20 µM) (schematic). Harvested timepoints were analyzed by immunoblotting. ( C ) PC3-Flag-AR/HA-PARP7 cells were pre-treated with transcription inhibitors triptolide (1 μM) or DRB (100 μM) for 1 h, followed by 6 h of androgen (2 nM R1881) treatment and analyzed by immunoblotting. ( D ) PC3-Flag-AR/HA-PARP7 cells were treated as described in (C). Cycloheximide (CHX) time course experiments were carried out under the indicated conditions and analyzed as described in A. Plots show mean ± SD from three independent experiments and protein half-lives. ( E ) Avi-tagged PARP7 was co-expressed in PC3M cells stably expressing HA-tagged AR (either wildtype [WT] or V582F mutant which targets the DNA binding domain [DBD MT]). Cells were treated with androgen (2 nM R1881, 6 h) and analyzed by immunoblotting.

Article Snippet: MG132, DRB, cycloheximide, triptolide, and enzalutamide (ENZ) were purchased from Cayman Chemical Company (Ann Arbor, MI, USA).

Techniques: Western Blot, Stable Transfection, Expressing, Mutagenesis, Binding 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