Journal: Journal of cancer research and therapeutics

Article Title: Triptolide exhibits dual anti-tumor effects through inhibiting autophagy and extracellular matrix activation in pancreatic cancer.

doi: 10.4103/jcrt.jcrt_186_24

Figure Lengend Snippet: Figure 1: Assessment of the impact of triptolide on the viability and proliferation of pancreatic cancer cells. The impact on pancreatic cancer cell lines was evaluated using the CCK‑8 assay after treatment with a concentration gradient of triptolide (a). The effect on proliferation in HPAC and a KPC‑derived cell line was assessed using the colony formation assay (b) Bar graph showing the number of clones for each cell line in b (c). In vivo experimental results demonstrated the inhibitory effects of triptolide on pancreatic orthotopic tumors and subcutaneous xenograft tumors (d). IHC images showing phospho‑Erk1/2‑ and phospho‑Histone H3‑positive cells in pancreatic orthotopic tumors treated with and without triptolide (left panels). The bar graph shows the quantification of phospho‑Erk1/2‑ and phospho‑Histone H3‑positive cells in the two groups (right panels) (e). Representative IHC images showing phospho‑ERK1/2‑ and phospho‑Histone H3‑positive cells in subcutaneous xenograft tumors treated with and without triptolide (left panels). Bar graph showing the quantification of phospho‑ERK1/2‑ and phospho‑Histone H3‑positive cells in the two groups (right panels) (f). IHC images demonstrating Ki‑67‑positive cells in pancreatic orthotopic and subcutaneous xenograft tumors treated with and without triptolide (left panels). The bar graph shows the quantification of Ki‑67‑positive cells in the two groups (right panels) (g). *P < 0.05, **P < 0.01, *** P < 0.001

Article Snippet: Total RNA was extracted from the Miapaca2 cells with or without triptolide treatment using TRIzol (15596026; Invitrogen), and RNA sequencing was performed on the BGI platform with 150‐bp pair‐end reads.

Techniques: CCK-8 Assay, Concentration Assay, Colony Assay, Clone Assay, In Vivo

Journal: Journal of cancer research and therapeutics

Article Title: Triptolide exhibits dual anti-tumor effects through inhibiting autophagy and extracellular matrix activation in pancreatic cancer.

doi: 10.4103/jcrt.jcrt_186_24

Figure Lengend Snippet: Figure 2: The impact of triptolide on apoptosis and cell cycle after treatment with different concentrations of triptolide. The impact on apoptosis of HPAC and the KPC‑derived cell line was assessed using FACS (a). A significant difference in the number of apoptotic cells was observed between the treatment and control groups (b). The impact of triptolide on the cell cycle in HPAC and a KPC‑derived cell line was assessed using FACS (c). Quantitative analysis showed a statistically significant difference in the number of G2/M phase of cells between the two groups (d). *P < 0.05, **P < 0.01, *** P < 0.001

Article Snippet: Total RNA was extracted from the Miapaca2 cells with or without triptolide treatment using TRIzol (15596026; Invitrogen), and RNA sequencing was performed on the BGI platform with 150‐bp pair‐end reads.

Techniques: Control

Journal: Journal of cancer research and therapeutics

Article Title: Triptolide exhibits dual anti-tumor effects through inhibiting autophagy and extracellular matrix activation in pancreatic cancer.

doi: 10.4103/jcrt.jcrt_186_24

Figure Lengend Snippet: Figure 3: The impact of triptolide on the extracellular stroma. Representative IHC images showing collagen and α‑SMA‑positive areas in orthotopic tumors constructed from KPC‑derived pancreatic cancer cells and subcutaneous xenografts constructed from HPAC cells treated with and without triptolide (scale bars: 50 μm) (a‑b). RT‑PCR revealed the relative expression levels of myCAF (ACTA2, CTGF, and MYL9) and iCAF (CFD) markers in human CAFs after co‑culture with HPAC (c) or Miapaca2 (d) cells with or without treatment with triptolide (20 nM). The expression levels of α‑SMA and COL1A1 in human CAFs after co‑culture with HPAC (e) or Miapaca2 (f) cells with or without treatment with triptolide (20 nM) after Western blotting. *P < 0.05, **P < 0.01, *** P < 0.001

Article Snippet: Total RNA was extracted from the Miapaca2 cells with or without triptolide treatment using TRIzol (15596026; Invitrogen), and RNA sequencing was performed on the BGI platform with 150‐bp pair‐end reads.

Techniques: Construct, Expressing, Western Blot

Journal: Journal of cancer research and therapeutics

Article Title: Triptolide exhibits dual anti-tumor effects through inhibiting autophagy and extracellular matrix activation in pancreatic cancer.

doi: 10.4103/jcrt.jcrt_186_24

Figure Lengend Snippet: Figure 4: Transcriptome analysis helped identify the mechanism by which triptolide suppresses tumor proliferation and stromal activation. Heatmap (left panel) and bar plot (right panel) showing significant differentially expressed genes (DEGs) by RNA sequencing in Miapaca2 cells treated with triptolide (20 nM) for 24 h (adjusted P value < 0.05; |log2FoldChange| >1) (a). Volcano plot showing significantly upregulated (red dots) and downregulated (blue dots) genes (b). BioPlanet pathway analysis revealed enriched pathways that were correlated with the attenuated TGF‑beta regulation of the ECM, senescence, and autophagy. The right panel shows the DEGs significantly enriched into these two pathways (c). Gene Set Enrichment Analysis (GSEA) using the Reactome Pathway term, Wikipathways term, and Gene Ontology (GO) term demonstrated significantly enriched pathways in the control group relative to the triptolide‑treated group (d)

Article Snippet: Total RNA was extracted from the Miapaca2 cells with or without triptolide treatment using TRIzol (15596026; Invitrogen), and RNA sequencing was performed on the BGI platform with 150‐bp pair‐end reads.

Techniques: Activation Assay, RNA Sequencing, Control

Journal: Journal of cancer research and therapeutics

Article Title: Triptolide exhibits dual anti-tumor effects through inhibiting autophagy and extracellular matrix activation in pancreatic cancer.

doi: 10.4103/jcrt.jcrt_186_24

Figure Lengend Snippet: Figure 5: The effects of triptolide on CXCL1 secretion, autophagy, and apoptosis in pancreatic cancer cells were observed via electron microscopy. Triptolide treatment significantly reduced the number of vesicles with double membranes in the treatment group compared to that in the control group, indicating the suppression of autophagy (a). Results of the Western blot analysis showing the expression levels of P62, Beclin‑1, ATG12, and LC3B‑I/II in HPAC and Miapaca2 cells with (10 nM and 20 nM) or without treatment with triptolide for 24 h (b). Results of the RT‑PCR analysis showing the relative expression of CXCL1 following the triptolide treatment (10 nM and 20 nM) for 24 h in the HPAC, Miapaca2, and KPC‑derived pancreatic cancer cells (c). Results of ELISA showing the concentration of CXCL1 in the cell supernatants of KPC‑derived pancreatic cancer cells following treatment with triptolide (10 nM and 20 nM) for 24 h (d). Results of ELISA showing the concentration of CXCL1 in the serum of mice with orthotopically implanted tumors using KPC‑derived pancreatic cancer cells with or without treatment with triptolide (e). Flow cytometry helped in detecting cell apoptosis in HPAC and Miapaca2 cells after triptolide treatment (20 nM) with or without CXCL1 (100 ng/mL) for 24 h (f). Results of the Western blot analysis showing the expression levels of P62 and LC3B‑I/II in HPAC and Miapaca2 cells with or without treatment with triptolide (20 nM) and CXCL1 (100 ng/mL) for 24 h (g). Results of the Western blot analysis showing the expression levels of mTOR, p‑mTOR, AKT, p‑AKT, ERK, and p‑ERK in HPAC and Miapaca2 cells with or without treatment with triptolide (10 nM and 20 nM) for 24 h (h). *P < 0.05, **P < 0.01, *** P < 0.001

Article Snippet: Total RNA was extracted from the Miapaca2 cells with or without triptolide treatment using TRIzol (15596026; Invitrogen), and RNA sequencing was performed on the BGI platform with 150‐bp pair‐end reads.

Techniques: Electron Microscopy, Control, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Concentration Assay, Flow Cytometry

Journal: Journal of cancer research and therapeutics

Article Title: Triptolide exhibits dual anti-tumor effects through inhibiting autophagy and extracellular matrix activation in pancreatic cancer.

doi: 10.4103/jcrt.jcrt_186_24

Figure Lengend Snippet: Figure 6: The impact of triptolide on TGF‑β‑1 secretion and activation of CAFs. The relative expression level of TGF‑β‑1 after treating HPAC, Miapaca2, and KPC‑derived pancreatic cancer cells with triptolide (10 nM and 20 nM) for 48 h (a). Results of the Western blot analysis showing the expression of TGF‑β‑1 in HPAC and Miapaca2 cells following triptolide treatment (10 nM and 20 nM) for 48 h (b). Results of ELISA showing the concentration of TGF‑β‑1 in the cell supernatants of the HPAC, Miapaca2, and KPC‑derived pancreatic cancer cells after 48 h of triptolide treatment (10 nM and 20 nM) (c). Results of ELISA showing the concentration of TGF‑β‑1 in the serum of mice with orthotopic xenografts using KPC‑derived pancreatic cancer cells and subcutaneous xenografts using HPAC cells with or without triptolide treatment (d). Results of the RT‑PCR analysis showing the expression levels of ACTA2, CTGF, and MYL9 in CAFs co‑cultured with HPAC and Miapaca2 cells with or without treatment with triptolide (20 nM) and TGF‑β‑1 (20 ng/mL) for 48 h (e). Results of the Western blot analysis showing the expression levels of α‑SMA and COL1A1 in CAFs co‑cultured with HPAC and Miapaca2 cells with or without treatment with triptolide (20 nM) and TGF‑β‑1 (20 ng/mL) for 48 h (f). *P < 0.05, **P < 0.01, *** P < 0.001

Article Snippet: Total RNA was extracted from the Miapaca2 cells with or without triptolide treatment using TRIzol (15596026; Invitrogen), and RNA sequencing was performed on the BGI platform with 150‐bp pair‐end reads.

Techniques: Activation Assay, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Concentration Assay

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:

Journal: IBRO Neuroscience Reports

Article Title: Neuroprotective effect of triptolide on neuronal inflammation in rats with mild brain injury

doi: 10.1016/j.ibneur.2024.05.007

Figure Lengend Snippet: Pathological changes in the hippocampus of mTBI rat. A.HE staining showed that the swelling and rupture of rat cells were decreased after triptolide treatment. B. number of neuron cells. Swelling and ruptured nerve cells can be seen in the image, as shown by the black arrow. ## P < 0.01 mTBI compared with the sham group in both day 3 and day 7; *P < 0.05, mTBI+TP compared with the mTBI group day 3 and day 7. scale bar 100 µm, n=3. C. NeuN immunostaning (red). D. NeuN positive cells.comparison between groups on day 7, and day 3 each group. ## P < 0.01 mTBI compared with the sham group in both day 3 and day 7; *P < 0.05, mTBI+TP compared with the mTBI group day 3 and day 7. Data presented as mean + SD. n=5. The *,and # , indicate statistically significant differences.

Article Snippet: The rats in the triptolide treatment group were intraperitoneally injected with 0.2 mg/kg triptolide ( ) for a week.TP (Med Chem Express, USA#39,836) was dissolved in DMSO at a concentration of 2 mg/mL and diluted with normal saline to a final concentration of 0.2 mg/ mL.

Techniques: Staining, Comparison

Journal: IBRO Neuroscience Reports

Article Title: Neuroprotective effect of triptolide on neuronal inflammation in rats with mild brain injury

doi: 10.1016/j.ibneur.2024.05.007

Figure Lengend Snippet: Inflammatory-related factors A. IL-1β, B. TNF-α and C. IL-10; changes after triptolide treatment. D&E individual raw data of each factors after 3 days and 7 days. Comparison between groups on day 3, comparison between groups on day 7, and day 3 and day 7 for each group. Data presented as mean + SD. The *, # , indicate statistically significant differences. IL-1β; mTBI Vs. Sham day3 ** P < 0.01, day 7 ** P < 0.01 , mTBI+TP Vs. mTBI; day3 ## P < 0.01, day 7 # P < 0.05, TNF-ɑ; mTBI Vs. Sham day3 ** P < 0.01, day 7 ** P < 0.01, mTBI+TP Vs. mTBI; day3 ## P < 0.01, day 7 ## P < 0.01, IL-10; mTBI Vs. Sham day3 * P < 0.05 , day 7 * P < 0.05, mTBI+TP Vs . mTBI; day3 ** P < 0.01, day 7 ** P < 0.01, n=10.

Article Snippet: The rats in the triptolide treatment group were intraperitoneally injected with 0.2 mg/kg triptolide ( ) for a week.TP (Med Chem Express, USA#39,836) was dissolved in DMSO at a concentration of 2 mg/mL and diluted with normal saline to a final concentration of 0.2 mg/ mL.

Techniques: Comparison

Journal: IBRO Neuroscience Reports

Article Title: Neuroprotective effect of triptolide on neuronal inflammation in rats with mild brain injury

doi: 10.1016/j.ibneur.2024.05.007

Figure Lengend Snippet: Triptolide downregulated IL-1β and NF-κB expressions. The relative mRNA expression was calculated using the formula 2– ΔΔ Ct. Comparison between groups on day 3, comparison between groups on day 7, and day 3 and day 7 for each group. Data presented as mean + SD. The indicate statistically significant differences. IL-1β; mTBI Vs. Sham day3 ** P < 0.01, day 7 ** P < 0.01 , mTBI+TP Vs. mTBI; day3 ## P < 0.01, day 7 # P < 0.05, NF-kB; mTBI Vs. Sham day3 ** P < 0.01, day 7 ** P < 0.01, mTBI+TP Vs. mTBI; day3 ## P < 0.01, day 7 ## P < 0.01, n=8.

Article Snippet: The rats in the triptolide treatment group were intraperitoneally injected with 0.2 mg/kg triptolide ( ) for a week.TP (Med Chem Express, USA#39,836) was dissolved in DMSO at a concentration of 2 mg/mL and diluted with normal saline to a final concentration of 0.2 mg/ mL.

Techniques: Expressing, Comparison

Journal: IBRO Neuroscience Reports

Article Title: Neuroprotective effect of triptolide on neuronal inflammation in rats with mild brain injury

doi: 10.1016/j.ibneur.2024.05.007

Figure Lengend Snippet: LC3B expression changes after triptolide treatment at different time points. (A): Representative LC3B positive (green) and DAPI (blue) merged image is shown. In the mTBI group, the positive expression index of autophagy protein LC3B was higher than both sham and mTBI + TP groups. The autophagy protein experimental group can be seen in the image. Scale bar = 50 μm. Immunofluorescence showed decreased expression of autophagy and aquaporin in rats treated with triptolide. (B): Comparison between groups on day 3, comparison between groups on day 7, and day 3 and day 7 for each group. Data presented as mean + SD. The *, #, indicate statistically significant differences. mTBI Vs. Sham day3 ** P < 0.01, day 7 ** P < 0.01 , mTBI+TP Vs. mTBI; day3 ## P < 0.01, day 7 ## P < 0.01 day 7, n=8. C. LC3B Western blot, D. Quantitative analysis of LC3B; mTBI Vs. Sham day3 ** P < 0.01, day 7 ** P < 0.01, mTBI+TP Vs. mTBI; day3 ## P < 0.01, day 7 ## P < 0.01 day 7, n=3.

Article Snippet: The rats in the triptolide treatment group were intraperitoneally injected with 0.2 mg/kg triptolide ( ) for a week.TP (Med Chem Express, USA#39,836) was dissolved in DMSO at a concentration of 2 mg/mL and diluted with normal saline to a final concentration of 0.2 mg/ mL.

Techniques: Expressing, Immunofluorescence, Comparison, Western Blot

Journal: IBRO Neuroscience Reports

Article Title: Neuroprotective effect of triptolide on neuronal inflammation in rats with mild brain injury

doi: 10.1016/j.ibneur.2024.05.007

Figure Lengend Snippet: AQP4 expression changes after triptolide treatment at different time points. (A): Representative AQP4 positive (green) and DAPI (blue) merged image is shown. In the mTBI group, the positive expression index of AQP4 was higher than the sham and mTBI + TP groups. The Aquaporin of an experimental group can be seen in the image. Scale bar = 50 μm. Immunofluorescence showed decreased expression of autophagy and aquaporin in rats treated with triptolide. (B): Comparison between groups on day 3, comparison between groups on day 7, and day 3 and day 7 for each group. Data presented as mean + SD, n=8. The *, #, indicate statistically significant differences. mTBI Vs. Sham day3 ** P < 0.01, day 7 ** P < 0.01 , mTBI+TP Vs. mTBI; day3 # P < 0.05, day 7 # P < 0.05 day 7, n=8. C. LC3B Western blot, D. Quantitative analysis of LC3B; mTBI Vs. Sham day3 ** P < 0.01, day 7 ** P < 0.01, mTBI+TP Vs. mTBI; day3 ## P < 0.01, day 7 ## P < 0.01 day 7, n=3.

Article Snippet: The rats in the triptolide treatment group were intraperitoneally injected with 0.2 mg/kg triptolide ( ) for a week.TP (Med Chem Express, USA#39,836) was dissolved in DMSO at a concentration of 2 mg/mL and diluted with normal saline to a final concentration of 0.2 mg/ mL.

Techniques: Expressing, Immunofluorescence, Comparison, Western Blot

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