Journal: PLOS One

Article Title: Down-regulation of circ0001361 induces apoptosis and suppresses the progression of glioma

doi: 10.1371/journal.pone.0343681

Figure Lengend Snippet: (A) MiRNAs targeted by circ0001361 were predicted. (B) Expression of hsa-miR-525-5p in glioma (Group A) and normal brain tissue (Group B) was shown. (C) Expression of hsa-miR-525-5p in glioma grade Ⅱ -Ⅳ was shown. (D) Prognostic significance of hsa-miR-525-5p was analyzed. (E) Potential binding sites between circ0001361 and hsa-miR-525-5p were predicted. (F) Circ0001361-hsa-miR-525-5p interaction was confirmed by dual-luciferase reporter assay.

Article Snippet: After 48 hours, the Firefly luciferase and Renilla Luciferase were detected using the Dual Luciferase Reporter Assay Kit (Vazyme, China).

Techniques: Expressing, Binding Assay, Luciferase, Reporter Assay

Journal: PLOS One

Article Title: Down-regulation of circ0001361 induces apoptosis and suppresses the progression of glioma

doi: 10.1371/journal.pone.0343681

Figure Lengend Snippet: (A) Genes targeted by hsa-miR-525-5p were predicted. (B) Correlation between hsa-miR-525-5p and MEIS1 was analyzed. (C) Prognostic significance of MEIS1 was analyzed. (D) Expression of MEIS1 in GBM, LGG and normal brain tissues was shown. (E) Expression of MEIS1 in glioma grade Ⅱ -Ⅳ was shown. (F) The relationship between MEIS1 and IDH mutation status was shown. (G) The relationship between MEIS1 and 1p/19q co−deletion status was shown. (H) Potential binding sites between hsa-miR-525-5p and MEIS1 were predicted. (I) Hsa-miR-525-5p and MEIS1 interaction was confirmed by dual-luciferase reporter assay.

Article Snippet: After 48 hours, the Firefly luciferase and Renilla Luciferase were detected using the Dual Luciferase Reporter Assay Kit (Vazyme, China).

Techniques: Expressing, Mutagenesis, Binding Assay, Luciferase, Reporter Assay

Journal: bioRxiv

Article Title: Epoxyeicosatrienoic acids and sEH inhibition prevent cardiac dysfunction in CVB3-induced myocarditis by positively regulating type I interferon signaling

doi: 10.1101/2023.02.03.527086

Figure Lengend Snippet: ( A ) Phosphorylation of IRF3 (on Ser396) in uninfected (CON) or CVB3-infected AC16 cells treated with 11,12-EET, 14,15-EET or TPPU (n=4). ( B ) Dimerization of IRF3 in uninfected (CON) or CVB3-infected AC16 cells treated with 11,12-EET, 14,15-EET or TPPU (n=4). ( C ) Nuclear translocation of IRF3 in uninfected (CON) or CVB3-infected AC16 cells treated with 11,12-EET, 14,15-EET or TPPU (n=4). GAPDH and LaminA/C were used as cytoplasm and nuclear protein loading controls. ( D ) Immunofluorescence images showing the impact of 14,15-EET and TPPU on the subcellular localization of IRF3 in uninfected (CON) or CVB3-infected AC16 cells; bars =50 μm. ( E ) Scheme showing the two main RNA sensor-induced IRF3 activation pathways. ( F-G ) AC16 cells that were uninfected (CON) or infected with CVB3 were treated with 11,12-EET, 14,15-EET or TPPU (n=3). Shown are changes in IRF3 phosphorylation following the siRNA-mediated down regulation of MDA5 (F), or TLR3 (G). ( H ) Activity of a IFNβ-luciferase reporter construct in HEK293 cells expressing either a control vector (vector), pcMDA5, pcMAVS, pcTBK1 , or pcIRF3-5D and treated with solvent (CON) or 14,15-EET (1 μM) for 24 hours. ( I ) Impact of the siRNA-mediated downregulation of TBK1 on the phosphorylation of IRF3 (on Ser396) in uninfected (CON) or CVB3-infected AC16 cells treated with 11,12-EET, 14,15-EET or TPPU (n=3). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: After 48 hours, transfected HEK293 cells were harvested in immunoprecipitation (IP) buffer containing 20 mM Tris (pH7.5), 150 mM NaCl, 1% Triton X-100 with freshly added PMSF, protease inhibitors and phosphatase inhibitors (Boster Biological Tech, Wuhan, China).

Techniques: Phospho-proteomics, Infection, Translocation Assay, Immunofluorescence, Activation Assay, Activity Assay, Luciferase, Construct, Expressing, Control, Plasmid Preparation, Solvent

Journal: bioRxiv

Article Title: Epoxyeicosatrienoic acids and sEH inhibition prevent cardiac dysfunction in CVB3-induced myocarditis by positively regulating type I interferon signaling

doi: 10.1101/2023.02.03.527086

Figure Lengend Snippet: ( A ) Impact of the siRNA-mediated downregulationof GSK3β on the phosphorylation of IRF3 (on Ser396) in uninfected (CON) or CVB3-infected AC16 cells treated with 11,12-EET, 14,15-EET or TPPU (n=4). ( B ) Impact of the EET antagonist; EEZE (1 μM) on the phosphorylation of GSK3β in AC16 cells treated with 11,12-EET, 14,15-EET or TPPU (n=4). ( C ) Co-immunoprecipitation of TBK1 with a GSK3β-Flag fusion protein from HEK293 cells treated with 14,15-EET (1 μM) or TPPU (10 μM) in the absence or presence of EEZE (n=3). ( D ) Impact of the wild-type GSK3β and the Y216F and Y216D GSK3β mutants on the phosphorylation of TBK1 in uninfected and CVB3-infected HEK293 cells (n=3). ( E-F ) Impact of GSK3β mutation alone and in combination with 14,15-EET on the phosphorylation of TBK1 in uninfected and CVB3-infected HEK293 cells (n=3-4). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: After 48 hours, transfected HEK293 cells were harvested in immunoprecipitation (IP) buffer containing 20 mM Tris (pH7.5), 150 mM NaCl, 1% Triton X-100 with freshly added PMSF, protease inhibitors and phosphatase inhibitors (Boster Biological Tech, Wuhan, China).

Techniques: Phospho-proteomics, Infection, Immunoprecipitation, Mutagenesis

Journal: Aging Cell

Article Title: Reduction of class I histone deacetylases ameliorates ER ‐mitochondria cross‐talk in Alzheimer's disease

doi: 10.1111/acel.13895

Figure Lengend Snippet: Tac effect on InsP3R1‐VDAC1 interaction and MERCS morphology in AβO‐treated cells. HT22 cells were pre‐treated for 1 h with Tac (10 μM) and then co‐incubated with 1 μM AβO for the remaining 23 h or treated solely with Tac for 24 h. Representative confocal images of in situ PLA signal (gray) indicating a physical interaction between InsP3R1 and VDAC1 (scale bar = 20 μm). The lower panels represent enlargements of the boxed areas in the upper panels. Quantification of number of PLA puncta in about 200–240 cells from 8 image stacks per condition (a). WT hippocampal neurons were pre‐treated for 1 h with Tac (0.5 μM) and then co‐incubated with 1 μM AβO for the remaining 23 h or treated solely with Tac for 24 h. NFAT transcriptional activity evaluated by luciferase reporter assay in hippocampal neurons (b) and HT22 cells (c). Data are the mean ± SEM of 4 independent experiments, run in triplicates to quadruplicates. Schematic representation of MERCS structural parameters: MERCS length and distance, mitochondrial perimeter and Feret's diameter (d). HT22 cells were pre‐treated for 1 h with Tac (10 μM) and then co‐incubated with 1 μM AβO for the remaining 23 h. Representative TEM images of mitochondria (in purple) in close contact with ER (in blue) (scale bar = 200 nm) in which distances ≤25 nm were considered as contacts (e). MERCS length (f) and distance (g), number of MERCS per mitochondria (h), mitochondrial area (i) and perimeter (j), Feret's diameter (k) and mitochondrial aspect ratio (l). Representative TEM images of mitochondrial cristae (in green) (scale bar = 200 nm) (m). Number of cristae per mitochondria (n), and cristae area (o). About 70–90 individual mitochondria from 10 randomly selected cells obtained from approximately 30 images were assessed in 26–38 independent TEM images. Statistical analysis: Kruskal–Wallis test followed by uncorrected Dunn's multiple comparisons test, one‐way ANOVA followed by uncorrected Fisher's LSD multiple comparison test; * p < 0.05; ** p < 0.01; *** p < 0.0001; **** p < 0.0001 when compared to control.

Article Snippet: NFAT luciferase reporter was obtained from Addgene.

Techniques: Incubation, In Situ, Activity Assay, Luciferase, Reporter Assay, Comparison, Control

Journal: Journal of Gastrointestinal Oncology

Article Title: Nestin overexpression reduces the sensitivity of gastric cancer cells to trastuzumab

doi: 10.21037/jgo-22-1048

Figure Lengend Snippet: Influence of Nestin on the activation of Nrf2/HO-1 and HER2/AKT/ERK1/2 of HER2-positive GC cells. The two cell lines received transfection by lentiviral-Nestin or lentiviral-empty, and then added to 10 μg/mL for 48 h. (A,B) Nestin mRNA level in MKN45 and NCI-N87 cells was detected by qRT-PCR. (C,D) WB was adopted to determine the protein level of Nestin, Nrf2, HO-1, NQO1, GST, AKT, ERK1/2, and HER2, in the MKN45 and NCI-N87 cells. (E,F) The cells were exposed to ARE luciferase reporter. 36 h post transfection, the luciferase activity was determined. Data are presented as the means ± SD of three independent experiments. *, P<0.05; **, P<0.01; ***, P<0.001. NTC, non-treated control; TRA, trastuzumab; TRA + NC, mice inoculated with cell infected with lentiviral-negative control were treated with TRA; TRA + Nestin, mice inoculated with cell infected with lentiviral-Nestin were treated with TRA; HO-1, hemeoxygenase-1; NQO1, NAD(P)H: quinone oxidoreductase-1; GST, glutathione S-transferase; AKT, protein kinase B; ERK1/2, extracellular regulated kinase 1/2; HER2, human epidermal growth factor receptor 2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ARE, antioxidant response element; GC, gastric cancer; qRT-PCR, quantitative real-time polymerase chain reaction; WB, Western blotting; SD, standard deviation.

Article Snippet: After a day of culture, the cells were treated with 200 ng of ARE luciferase reporter plasmids using OriGene transfection reagent (Company, City, Country).

Techniques: Activation Assay, Transfection, Quantitative RT-PCR, Luciferase, Activity Assay, Control, Infection, Negative Control, Real-time Polymerase Chain Reaction, Western Blot, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: Discovery of small molecules and a druggable groove that regulate DNA binding and release of the AP-1 transcription factor ΔFOSB

doi: 10.1016/j.jbc.2025.111080

Figure Lengend Snippet: Cellular activity of JPC0661 . A , cell viability of JPC0661 comm and HTS10307 assessed in the range of 0 to 20 μM after 24 h ( top ) and 72 h ( bottom ) in neural progenitor cells (NPCs) using the CellTiter-Glo viability assay. Cell viability measures are normalized to the negative control containing no compound but 0.6% DMSO. Dotted lines indicate 100% cell viability. The mean of n replicates is shown for 24 h (n = 3) and 72 h (n = 2) with error bars indicating the SEM. B , stability of JPC0661 comm and HTS10307 in mouse liver microsomes. C and D , effects of JPC0661 comm (0–100 μM; ( C ) and JPC0661 (0–100 μM; ( D ) on AP-1-driven luciferase activity in AP-1-luc HEK293 cells. The dose-dependent activation of the AP-1-driven luciferase reporter was measured based on changes in the luciferase signal and expressed as relative fluorescence units (RFUs). Each compound was tested twice (n = 4 wells per experiment; total n = 6–8 wells for JPC0661 comm and n = 7–8 wells for JPC0661 per concentration), with results normalized to the luciferase signals of blank wells from corresponding experiments (n = 8 wells). Nonlinear regression using a three-parameter model was applied to fit the luciferase signal and calculate IC 50 values, which are reported with a 95% confidence interval (CI), and data points are presented as the mean ± SEM. E , effect of serially diluted JPC0661 (0.003–100 μM) on the viability of AP-1-luc HEK293 cells using the Celltiter-Glo viability assay (2 h). Cell viability was normalized to the DMSO control, which contained 0.5% DMSO but no compound (n = 7 wells). Data points are presented as the mean ± SEM, with a total n = 10 to 12 wells per concentration. F , effect of JPC0661 (at doses 12.5, 25, and 50 μM) on the viability of Neuro 2A cells (72 h) using the Celltiter-Glo viability assay to assess potential toxicity in mouse neuronal cells. Cell viability was normalized to the DMSO control, which contained 0.5% DMSO but no compound (n = 8 wells). Data points are presented as the mean ± SEM, with a total n = 12 wells per concentration. AP-1, activator protein 1; DMSO, dimethyl sulfoxide.

Article Snippet: The AP-1-luciferase reporter HEK293 cell line was obtained from BPS Bioscience and maintained in growth medium 1B (BPS Bioscience) supplemented with 10% fetal bovine serum (FBS; Invitrogen) and 1% penicillin–streptomycin (Hyclone) under standard incubation conditions of 37 °C with 5% CO 2 .

Techniques: Activity Assay, Viability Assay, Negative Control, Luciferase, Activation Assay, Fluorescence, Concentration Assay, Control

Journal: The Journal of Biological Chemistry

Article Title: Discovery of small molecules and a druggable groove that regulate DNA binding and release of the AP-1 transcription factor ΔFOSB

doi: 10.1016/j.jbc.2025.111080

Figure Lengend Snippet: Validation of JPC0661 as an inhibitor of ΔFOSB . A , a panel of JPC0661 analogs was designed to probe the role of the sulfonic acid group and the amino-pyrazolone group. B , the table summarizing the JPC0661 analogs tested in AP-1-reporter assays using AP-1-luc HEK293 cells, yielding cell-based IC 50 values and FP-DRC assays. Lower IC 50 values are marked with more plus signs (+) and indicate higher activity. The plots from the FP-DRC and cell-based DRC assays for these compounds are shown in . C , ΔFOSB–JUND bZIP incubated with compounds (0.5 mM) with 100 μM diamide (“ ox ,” oxidized) or without diamide (“ red ,” reduced) and assessed by SDS-PAGE (with or without reducing agent in the loading buffer). D , ΔFOSB–JUND bZIP protein incubated with compounds (0.5 mM) with no diamide (“ red ”) or protein alone (no compound) incubated with diamide (“ ox ”) as a control and then assessed by SDS-PAGE (with or without reducing agent in the loading buffer). In ( C ) and ( D ), “cntrl” denotes the ΔFOSB–JUND bZIP protein in the absence of compound. AP-1, activator protein 1; bZIP, basic leucine zipper; FP-DRC, fluorescence polarization–dose–response curve; HEK293, human embryonic kidney 293 cell line.

Article Snippet: The AP-1-luciferase reporter HEK293 cell line was obtained from BPS Bioscience and maintained in growth medium 1B (BPS Bioscience) supplemented with 10% fetal bovine serum (FBS; Invitrogen) and 1% penicillin–streptomycin (Hyclone) under standard incubation conditions of 37 °C with 5% CO 2 .

Techniques: Biomarker Discovery, Activity Assay, Incubation, SDS Page, Control, Fluorescence