Journal: eLife

Article Title: P2Y1 purinergic receptor identified as a diabetes target in a small-molecule screen to reverse circadian β-cell failure

doi: 10.7554/eLife.75132

Figure Lengend Snippet: ( A ) Significant Z-scores (>3 standard deviations) and fold changes (>1.25-fold increase) for all 2640 screened compounds, with hit compounds indicated in blue. ( B ) Top 12 hit compounds identified from screen with a fold increase > 1.25 and a Z-score > 3, which were selected for further analysis. Known functions and published molecular pathways targeted by these compounds are indicated. ( C ) Model of potential mechanisms of action of the top 12 hit compounds to affect insulin secretion in the β-cell. ( D ) Glucose-responsive insulin secretion by ELISA at 2 mM and 20 mM glucose in WT mouse islets following exposure to four lead candidate compounds (n = 3–11 mice/compound). ( E ) Ivermectin (IVM) dose-response curve (n = 6–8 experimental repeats/dose), ranging from 0.078 µM to 80 µM IVM, in insulin-NanoLuciferase-expressing Beta-TC-6 cells. Shaded area represents 95% confidence intervals for the LOESS curve. All values represent mean ± SEM. **p<0.01, ***p<0.001.

Article Snippet: Chemical compound, drug , Ivermectin , Tocris , 1260 , .

Techniques: Enzyme-linked Immunosorbent Assay, Expressing

Journal: eLife

Article Title: P2Y1 purinergic receptor identified as a diabetes target in a small-molecule screen to reverse circadian β-cell failure

doi: 10.7554/eLife.75132

Figure Lengend Snippet: ( A ) mRNA abundance (transcripts per million [TPM]) in WT β-cells (left), DESeq2-adjusted p-values from differential expression analysis in Bmal1 -/- versus WT β-cells (middle left), fold change in expression in Bmal1 -/- versus WT β-cells (middle right), and presence or absence of an annotated BMAL1 binding site near genes of putative ivermectin (IVM) targets (right). ( B ) Rhythmic expression of P2ry1 gene in synchronized pseudoislets from WT Beta-TC-6 cells as assessed by quantitative real-time PCR (n = 3) (false discovery rate (FDR)-adjusted p-value<0.05). ( C ) Uniform manifold approximation and projection (UMAP) clustering analysis of single-cell expression values in single human islet cells isolated from type 2 diabetic and healthy subjects highlights distinct transcriptional profiles of β, α, δ, and γ cells marked by high levels of insulin ( INS ), glucagon ( GCG ), somatostatin ( SST ), or pancreatic polypeptide ( PPY ) mRNA, respectively. P2RY1 expression is enriched in β and δ cells, and grossly excluded from α and γ cells.

Article Snippet: Chemical compound, drug , Ivermectin , Tocris , 1260 , .

Techniques: Quantitative Proteomics, Expressing, Binding Assay, Real-time Polymerase Chain Reaction, Isolation

Journal: eLife

Article Title: P2Y1 purinergic receptor identified as a diabetes target in a small-molecule screen to reverse circadian β-cell failure

doi: 10.7554/eLife.75132

Figure Lengend Snippet:

Article Snippet: Chemical compound, drug , Ivermectin , Tocris , 1260 , .

Techniques: Mutagenesis, Double Knockout, Isolation, Expressing, Recombinant, CRISPR, Plasmid Preparation, Drug discovery, Luciferase, Enzyme-linked Immunosorbent Assay, Reverse Transcription, SYBR Green Assay, Bradford Protein Assay, Sequencing, Software, Modification, Protease Inhibitor

Journal: Journal of biomedical science

Article Title: Targeting the G-quadruplex as a novel strategy for developing antibiotics against hypervirulent drug-resistant Staphylococcus aureus.

doi: 10.1186/s12929-024-01109-3

Figure Lengend Snippet: Fig. 7 NMM stabilizes the G4-motif in PmraZ promoter to inhibit transcription/translation. A, B Three constructs showing promoter-less mCherry empty vector (EV) (A I), the recombinant fusion protein, rMraZ-His6-mCherry expression under PgapA and PmraZ promoters (A II–III) were transformed in E. coli DH5α showing the differential expression of rMraZ-His6-mCherry fusion protein (B I–III). C, D Qualitative and quantitative assessment of differential expression of rMraZ-His6-mCherry fusion protein without or with 5 µM NMM using qualitative confocal fluorescence microscopy (C I–III); and quantitative fluorometric estimation of red fluorescent signal intensity of rMraZ-His6-mCherry fusion protein showing that the NMM inhibits the rMraZ-His6-mCherry fusion protein expression due to the inhibition of bacterial coupled transcription/translation (D). E DNA templates for IVT reactions showing T7 promoter with G4-motif (WT PmraZ_G4_3) (lane 1) and T7 promoter without G4-motif (lane 2) tagged with mraZ-his6-stop. F, G Western blot and immunodetection of recombinant MraZ-His6 protein using Anti-His antibody showed the NMM concentration-dependent inhibition of coupled transcription/translation in case of T7 promoter with G4-motif (WT PmraZ_G4_3) (F), while such inhibition was absent where T7 promoter is devoid of G4-motif (G)

Article Snippet: Immunodetection of recombinant MraZ-His6 protein of IVT reactions without or with NMM was carried out with a primary mouse-monoclonal anti-His antibody (Santa Cruz Biotechnology, USA).

Techniques: Construct, Plasmid Preparation, Recombinant, Expressing, Transformation Assay, Quantitative Proteomics, Fluorescence, Microscopy, Inhibition, Western Blot, Immunodetection, Concentration Assay

Journal: International Journal of Medical Sciences

Article Title: Ivermectin induces cell cycle arrest and caspase-dependent apoptosis in human urothelial carcinoma cells

doi: 10.7150/ijms.76623

Figure Lengend Snippet: Ivermectin induces apoptosis in human urothelial carcinoma cells. RT4 cells were treated with ivermectin, (A) and early apoptotic cells were examined via flow cytometry. (B) Caspase-3, -8, -9, PARP, Bid, and Bcl-xL protein expression was assessed using western blotting in cells treated with ivermectin. (C) MMP was evaluated via flow cytometry in RT4 cells after treatment with ivermectin. DMSO was used as negative control. Three independent experiments each were conducted for flow cytometry and western blotting; Western blots and flow cytometry dot plots of a representative experiment are shown. *Compared with the control group. ** p < 0.01, *** p < 0.001.

Article Snippet: To investigate the mechanisms underlying ivermectin-mediated apoptosis, the activation of caspase-3, -8, -9 (#9662, #9746, #9502, Cell Signaling; with 1:1000 dilution), Bid (GTX60429, 1:1000, GeneTex, Hsinchu City, Taiwan), Bcl-xL (#2762, Cell Signaling) and poly (ADP-ribose) polymerase (PARP; #9542, Cell Signaling; with 1:1000 dilution) were assessed using western blotting.

Techniques: Flow Cytometry, Expressing, Western Blot, Negative Control, Control

Journal: International Journal of Medical Sciences

Article Title: Ivermectin induces cell cycle arrest and caspase-dependent apoptosis in human urothelial carcinoma cells

doi: 10.7150/ijms.76623

Figure Lengend Snippet: Ivermectin-induced apoptosis in human urothelial carcinoma cells was caspase-dependent. RT4 cells were pre-incubated with Z-VAD-FMK before ivermectin treatment, (A) caspase-3 and PARP expression was examined using western blotting, and (B) early apoptotic cells were quantified via flow cytometry. (C) Total cellular viability was measured via CCK-8 assay. DMSO was used as negative control. Three independent experiments each were conducted for flow cytometry and western blotting; western blots and flow cytometry dot plots of a representative experiment are shown. *Compared with the control group, and # compared with the ivermectin treatment group. *** and ### p < 0.001.

Article Snippet: To investigate the mechanisms underlying ivermectin-mediated apoptosis, the activation of caspase-3, -8, -9 (#9662, #9746, #9502, Cell Signaling; with 1:1000 dilution), Bid (GTX60429, 1:1000, GeneTex, Hsinchu City, Taiwan), Bcl-xL (#2762, Cell Signaling) and poly (ADP-ribose) polymerase (PARP; #9542, Cell Signaling; with 1:1000 dilution) were assessed using western blotting.

Techniques: Incubation, Expressing, Western Blot, Flow Cytometry, CCK-8 Assay, Negative Control, Control

Journal: International Journal of Medical Sciences

Article Title: Ivermectin induces cell cycle arrest and caspase-dependent apoptosis in human urothelial carcinoma cells

doi: 10.7150/ijms.76623

Figure Lengend Snippet: Ivermectin-mediated apoptosis through suppression of JNK signaling. (A) RT4 cells were treated with ivermectin, and p38, ERK, and JNK expressions were assessed using western blotting. (B) PD98059 and (C) SP600125 were used to inhibit ERK or JNK activation, and ERK and JNK expression and activation were confirmed using western blotting. Total apoptotic cells and total cell survival were evaluated via flow cytometry and CCK-8 assay. DMSO was used as negative control. Three independent experiments each were conducted for flow cytometry and western blotting; Western blots and flow cytometry of a representative experiment are shown. *Compared with the control group, and # compared with the ivermectin treated group. *** and ### p < 0.001.

Article Snippet: To investigate the mechanisms underlying ivermectin-mediated apoptosis, the activation of caspase-3, -8, -9 (#9662, #9746, #9502, Cell Signaling; with 1:1000 dilution), Bid (GTX60429, 1:1000, GeneTex, Hsinchu City, Taiwan), Bcl-xL (#2762, Cell Signaling) and poly (ADP-ribose) polymerase (PARP; #9542, Cell Signaling; with 1:1000 dilution) were assessed using western blotting.

Techniques: Western Blot, Activation Assay, Expressing, Flow Cytometry, CCK-8 Assay, Negative Control, Control