Journal: Cell reports

Article Title: A Role for FACT in RNA Polymerase II Promoter-Proximal Pausing.

doi: 10.1016/j.celrep.2019.05.099

Figure Lengend Snippet: Figure 4. Paused Pol II Half-Life (t1/2) Decreases in FACT-Depleted S2 Cells (A–D) Chromatin from cells that had been treated with control dsRNA targeting EGFP or dsRNA targeting SSRP1 were incubated with 500-mM triptolide for 5, 10, 15, or 30 min or with 2% DMSO as control and subjected to ChIP-nexus using anti-Rpb3 antibodies. For each promoter where a typical Pol II ChIP-nexus footprint was observed (distance between positive and negative strand peak < 50 bp, position of Pol II footprint < 150 bp downstream of the TSS), spike-in normalized Pol II signal in a 51-bp window centered on the midpoint between paused Pol II positive and negative summits was determined, and the half-life of paused Pol II was calculated based on an exponential decay model. (A) Pol II in the pausing window at 18w as a function of time after addition of triptolide, in cells treated with EGFP (red) or SSRP1 (blue) dsRNAs. (B) Boxplot showing decreased paused Pol II half-life following SSRP1 depletion. p value was calculated with a two-sample Wilcoxon test, n = 998. (C) Heatmaps of Pol II ChIP-nexus data at various times after triptolide addition in EGFP and SSRP1 dsRNA-treated cells. (D) Boxplot showing fold change in pause half-life across highly paused (n = 411), moderately paused (n = 358), and lowly paused (n = 229) genes. p values were calculated using Wilcoxon rank sum test of pairwise comparisons. (E) A model for FACT function in promoter-proximal pausing and transcription-coupled histone modifications. In control cells (top panel), the +1 nucleosome is stabilized by FACT (Ramachandran et al., 2017). The +1 nucleosome in turn helps to maintain Pol II in the vicinity of the promoter-proximal pause, and tran- scription-coupled histone modifications H3K4me3 and H3K36me3 are normally deposited. Upon depletion of FACT, the +1 nucleosome is destabilized, and Pol II spends less time at the promoter-proximal pause. Hence, methyltransferases associated with elongating Pol II are allowed less time to place marks, leading to a broadening of H3K4me3 and relative depletion of H3K36me3 from more 50 portions of genes.

Article Snippet: To control for the loss of Pol II ChIP signal after triptolide treatment, a spike-in control was prepared by incubating human chromatin extracts from GM12878 cells with a 1:1 mixture of Dynabeads Protein A and Dynabeads Protein G coupled to antibodies against human Pol II (N20, Santa Cruz) followed by washing with nexus washing buffers A to D. A fixed amount of Dynabeads and human Pol II ChIP mixture was then spiked into each S2 ChIP-nexus experiment.

Techniques: Control, Incubation

Journal: Molecular Medicine Reports

Article Title: Triptolide inhibits cell proliferation and tumorigenicity of human neuroblastoma cells

doi: 10.3892/mmr.2014.2814

Figure Lengend Snippet: TPL inhibited neuroblastoma cell growth and viability. (A) BE(2)-C cells were treated with 5, 10, 25, 50 and 100 nM TPL for 24 h. DMSO was used as a control. Cell viability was assessed by a CCK-8 assay. (B) BE(2)-C cell numbers from panel C were counted using the TC10™ Automated Cell Counter.(C) Morphologic examination of BE (2)-C cells treated with 25 nM triptolide for the indicated times (0, 24 or 48 h). Scale bar, 100 μ m. Each value represents the average obtained from three independent experiments. Data are presented as the mean ± standard deviation. * P<0.05 and ** P<0.01, compared with control. CCK-8, cell counting kit-8; TPL, triptolide; DMSO, dimethyl sufoxide.

Article Snippet: After treatment with triptolide (25 nM) for 24 h, cells were collected by centrifugation at 211 × g for 5 min, washed with ice-cold PBS, fixed with 70% ethanol, stained with 20 μ g/ml propidium iodide (Invitrogen) and analyzed by flow cytometry (BD FACSVerseTM, BD BioSciences, Franklin Lakes, NJ, USA).

Techniques: Control, CCK-8 Assay, Standard Deviation, Cell Counting

Journal: Molecular Medicine Reports

Article Title: Triptolide inhibits cell proliferation and tumorigenicity of human neuroblastoma cells

doi: 10.3892/mmr.2014.2814

Figure Lengend Snippet: BrdU immunofluorescence staining assay. (A) Immunofluorescence staining of Brdu in BE(2)-C cells treated with triptolide (25 and 50 nM) for 24 h. Scale bars, 25 μ m. (B) The percentage of BrdU positive cells from panel A was calculated. Each value represents the average obtained from three independent experiments. Data are presented as the mean ± standard deviation. * P<0.05 and ** P<0.01, compared with control. BrdU, 5-bromo-2-deoxyuridine; DMSO, dimethyl sulfoxide.

Article Snippet: After treatment with triptolide (25 nM) for 24 h, cells were collected by centrifugation at 211 × g for 5 min, washed with ice-cold PBS, fixed with 70% ethanol, stained with 20 μ g/ml propidium iodide (Invitrogen) and analyzed by flow cytometry (BD FACSVerseTM, BD BioSciences, Franklin Lakes, NJ, USA).

Techniques: Immunofluorescence, Staining, Standard Deviation, Control

Journal: Molecular Medicine Reports

Article Title: Triptolide inhibits cell proliferation and tumorigenicity of human neuroblastoma cells

doi: 10.3892/mmr.2014.2814

Figure Lengend Snippet: Triptolide induced neuroblastoma cell cycle arrest in the S phase. (A) BE(2)-C cells were either treated with DMSO or 25 nM triptolide for 24 h. Cells were harvested, fixed with ethanol and stained with propidium iodide. DNA content was determined by flow cytometry. (B) Analysis of cell cycle phase percentage in BE(2)-C cells from panel A. Each column represents the average obtained from three independent experiments. Data are presented as the mean ± standard deviation. * P<0.05 and ** P<0.01, compared with control. DMSO, dimethyl sulfoxide.

Article Snippet: After treatment with triptolide (25 nM) for 24 h, cells were collected by centrifugation at 211 × g for 5 min, washed with ice-cold PBS, fixed with 70% ethanol, stained with 20 μ g/ml propidium iodide (Invitrogen) and analyzed by flow cytometry (BD FACSVerseTM, BD BioSciences, Franklin Lakes, NJ, USA).

Techniques: Staining, Flow Cytometry, Standard Deviation, Control

Journal: Molecular Medicine Reports

Article Title: Triptolide inhibits cell proliferation and tumorigenicity of human neuroblastoma cells

doi: 10.3892/mmr.2014.2814

Figure Lengend Snippet: Triptolide induced neuroblastoma cell apoptosis through caspase-9/caspase-3 pathway activation. Analysis of the percentage of (A) dead and (B) apoptotic BE(2)-C cells. BE(2)-C cells were treated with 25nM triptolide for 24 h, and cell death and apoptosis were determined by trypan blue dye and Annexin V-fluorescein isothiocyanate kit, respectively. DMSO was used as a control. (C) mRNA expression levels of caspase-3 and caspase-9 in BE(2)-C cells treated with DMSO or triptolide were determined by RT-qPCR analysis. Data represent the average obtained from three independent experiments. Data are presented as the mean ± standard deviation. * P<0.05 and ** P<0.01, compared with control. RT-qPCR, reverse transcription-quantitative polymerase chain reaction; DMSO, dimethyl sulfoxide.

Article Snippet: After treatment with triptolide (25 nM) for 24 h, cells were collected by centrifugation at 211 × g for 5 min, washed with ice-cold PBS, fixed with 70% ethanol, stained with 20 μ g/ml propidium iodide (Invitrogen) and analyzed by flow cytometry (BD FACSVerseTM, BD BioSciences, Franklin Lakes, NJ, USA).

Techniques: Activation Assay, Control, Expressing, Quantitative RT-PCR, Standard Deviation, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Molecular Medicine Reports

Article Title: Triptolide inhibits cell proliferation and tumorigenicity of human neuroblastoma cells

doi: 10.3892/mmr.2014.2814

Figure Lengend Snippet: Triptolide suppressed BE (2)-C cells colony-forming capability. (A) Soft agar clonogenic assay of BE(2)-C cells was performed after treatment with triptolide. After 14 days of culture, images of colonies (larger than 1.0 mm or containing more than 150 cells) were captured. Scale bars, 50 μ m. (B) Analysis of colony formation numbers from panel A was performed. Cells were counted from at least five randomly selected fields. Data are presented as the mean ± standard deviation. * P<0.05 and ** P<0.01, compared with control. DMSO, dimethyl sulfoxide.

Article Snippet: After treatment with triptolide (25 nM) for 24 h, cells were collected by centrifugation at 211 × g for 5 min, washed with ice-cold PBS, fixed with 70% ethanol, stained with 20 μ g/ml propidium iodide (Invitrogen) and analyzed by flow cytometry (BD FACSVerseTM, BD BioSciences, Franklin Lakes, NJ, USA).

Techniques: Clonogenic Assay, Standard Deviation, Control

Journal: Molecular Medicine Reports

Article Title: Triptolide inhibits cell proliferation and tumorigenicity of human neuroblastoma cells

doi: 10.3892/mmr.2014.2814

Figure Lengend Snippet: Triptolide inhibited tumor growth and development in the xenograft model. BE(2)-C cells (1×10 6 ) were injected subcutaneously into the flanks of NOD/SCID mice. After tumor formation (approximately two weeks), mice were injected with DMSO or triptolide (0.4 mg/kg) daily for seven days. (A) Images of tumors dissected from NOD/SCID mice after treatment with triptolide or DMSO. (B) Xenograft tumors were measured daily, after treatment with triptolide or DMSO, by calipers. (C) Scatter plot of xenograft tumor weight with horizontal lines indicated the mean in each group. (D) The average body weight of NOD/SCID mice was monitored daily after treatment with triptolide or DMSO. Data are presented as the mean ± standard deviation. * P<0.05 and ** P<0.01, compared with control. DMSO, dimethyl sulfoxide; NOD mice, non-obese diabetic mice; SCID mice, severe combined immunodeficiency mice.

Article Snippet: After treatment with triptolide (25 nM) for 24 h, cells were collected by centrifugation at 211 × g for 5 min, washed with ice-cold PBS, fixed with 70% ethanol, stained with 20 μ g/ml propidium iodide (Invitrogen) and analyzed by flow cytometry (BD FACSVerseTM, BD BioSciences, Franklin Lakes, NJ, USA).

Techniques: Injection, Standard Deviation, Control

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Advances in nanomedicine and delivery systems for gastric cancer research

doi: 10.3389/fbioe.2025.1565999

Figure Lengend Snippet: Promising areas for nanomedicine development in gastric cancer.

Article Snippet: Biomimetic Nanoparticles , , PTX, Triptolide , NPs , RP (P/T).

Techniques:

Journal: Nucleic Acids Research

Article Title: ADAR3 modulates neuronal differentiation and regulates mRNA stability and translation

doi: 10.1093/nar/gkae753

Figure Lengend Snippet: ADAR3 influences mRNA stability. ( A ) Scatterplot comparing mRNA decay rates in N2A-C and N2A-A3 cells following treatment with triptolide for 8 h. For each transcript and each cell line, the ratio between the normalized abundances at 0 and at 8 h was calculated and expressed as a log2 fold change. The plot shows the average ratio of the log2 fold change ( n = 4) in each cell line. The x-axis shows the value for N2A-C and the y-axis shows the values for N2A-A3 cells. Only the top 10% of stabilized or destabilized transcripts were considered significant. Destabilized transcripts in N2A-A3 cells are shown in red and stabilized transcripts in blue. See Methods for details. ( B ) Gene ontology enrichment test (GO: Biological processes) of destabilized (right) and stabilized (left) transcripts. The x-axis indicates Fold Enrichment, color signifies –log 10 False Discovery Rate (FDR) and the size of the dot indicates the number of genes in the dataset that belong to the pathway. ( C ) Scatter plot comparing changes in mRNA stability (x-axis) with changes in mRNA abundance (y-axis). The plot includes all transcripts with available mRNA stability data in the triptolide experiment, except those showing increased abundance after triptolide treatment. Differentially expressed transcripts are shown in blue. Pearson's correlations are shown in the figure. n = 6304 transcripts. ( D ) Scatter plot as in C including only the mRNAs that showed extreme changes in mRNA stability in the triptolide experiment (stability residual > 0.5 and stability residual < –0.5). Differentially expressed transcripts (FDR < 0.05) are shown in blue. Pearson's correlation is shown in the figure.

Article Snippet: For triptolide treatment, cells were seeded at 300 000 cells per well in a 12-well plate and treated with 1 μM triptolide (Tocris) 24 h after seeding.

Techniques: