pkcβ inhibitor  (Santa Cruz Biotechnology)

Journal: Nature Communications

Article Title: Spatial transcriptomics of a parasitic flatworm provides a molecular map of drug targets and drug resistance genes

doi: 10.1038/s41467-024-53215-3

Figure Lengend Snippet: a Flow chart showing gene numbers present in each step of our drug repurposing analysis. Eleven potential targets of 24 drugs were predicted in the tegument and ten targets of 37 drugs in the gut. b SMART analysis and alignment of catalytic domains of human and liver fluke PKCβ. The domain composition (C2 regulatory domain in front of the kinase domain) classifies both as conventional PKCs . The catalytic domain of human PKCβ, which is bound by ruboxistaurin, shows 73.36% identity to the Fasciola ortholog. The conserved ATP binding site (bold) and the activation loop residues (underlined) are marked. The main residues involved in ruboxistaurin binding obtained for the human sequence are Lys349, Gly350 and Lys467 (gray). c Left: Heatmap showing the average expression of different protein kinase C (PKC) genes per cluster (mean of UMI counts, normalized and scaled). Right: Heatmap showing the average spot expression of those genes in the whole dataset (log1p normalized counts). Red rectangle marks PKCβ with tegumental expression. Please note: While the spatial plot is shown for only one representative section, the heatmap includes expression data from all four tissue sections in the dataset. Source data are provided as a Source Data file. d Spatial projection only (left) and overlay with H&E-stained tissue section (right) showing expression of PKCβ (D915_006901). Several positive spots can be seen along the tegument of the parasite. Mehlis’ gland is not contained in this tissue section. For spatial projections of other PKCs, see Supplementary Fig. . Expression level encoded by color (gray = low, red = high). e – g Adult flukes were treated for 72 h with different concentrations of the PCKβ isoform-specific inhibitor ruboxistaurin (20–100 µM) or triclabendazole as positive control. Motility was assessed every 24 h. Control worms were treated with the inhibitor solvent DMSO. Representative images are shown in ( e ) and motility scores for all time points and concentrations in ( f and g ) (score 3 = normal, 2 = reduced, 1 = severely reduced, 0 = no motility). See also Supplementary Movies and . Data represent the mean ± SEM of n = 4 (triclabendazole at 20 µM) or n = 6 flukes (others) from 2 (triclabendazole at 20 µM) or 3 independent experiments (others) with 2 worms per condition and experiment. Significant differences to controls are indicated with * p = 0.0333 and ** p = 0.0022 (two-sided Mann-Whitney U test). Scale bars correspond to 5 mm.

Article Snippet: The anthelminthic activity of the PKCβ inhibitor ruboxistaurin (LY333531, S7663, Selleckchem) against adult stages of F. hepatica was assessed in vitro using different inhibitor concentrations (20, 50, or 100 μM).

Techniques: Binding Assay, Activation Assay, Sequencing, Expressing, Staining, Positive Control, Control, Solvent, MANN-WHITNEY

Journal: bioRxiv

Article Title: B lymphocytes acquire myeloid and autoimmune phenotypes via the downregulation of lymphocyte-specific protein-1

doi: 10.1101/2024.06.28.600734

Figure Lengend Snippet: a, Venn diagram displaying transcription factors (TFs) that have a high potential to regulate the 369 upregulated DEGs in Lsp1 -/- B cells with or without αIgM or LPS treatment (left panel). The bar graph on the right depicts the numbers of C/EBP target genes among the DEGs in Lsp1 -/- B cells. b, PPI network representing the interactions between C/EBPα/β and the 31 C/EBP target genes involved hematopoietic cell lineage markers, cytokines/chemokines, and phagocytosis. The color of the nodes indicates the fold change in the genes affected by Lsp1 deficiency. c, d, Quantitative RT‒PCR analysis of Cebpa and Cebpb mRNA expression (n=4∼7; c ) and immunoblotting for C/EBPβ protein expression ( d ). Lsp1 -/- versus WT B cells were stimulated with αIgM or LPS for 4 hours ( c ) or for 24 hours ( d ). e, f, Lsp1 -/- B cells were transduced with control or Cebpb shRNAs, and the cells were then stimulated with αIgM or LPS for 24 hours for C/EBPβ immunoblotting ( e ) and MPO and TNF-α ELISAs (n=4; f ). g, Control or Cebpb shRNA-treated Lsp1 -/- B cells were stimulated with αIgM or LPS for 5 days, after which Blimp1, IRF4, and Xbp1 expression was determined via immunoblot assay. h, Immunoblots of phosphorylated PKCβ (pPKCβ) in Lsp1 -/- versus WT B cells. Splenic B cells were stimulated with αIgM or LPS for 5 minutes. i, j, Effects of PKCβ inhibition on C/EBPβ and IL-1β expression in Lsp1 -/- B cells stimulated with αIgM or LPS. ( i ) Lsp1 -/- B cells were pretreated with the PKCβ inhibitor ruboxistaurin (4 μM) for 1 hour and stimulated with αIgM or LPS for 24 hours. ( j) Lsp1 -/- B cells were stimulated with αIgM or LPS for 24 hours and then electroporated with control or Prkcb siRNA for an additional 24 hours. The expression levels of C/EBPβ, its target gene IL-1β, and PKCβ were determined via an immunoblot assay. k, Proposed model for the dual regulation of B-cell functions by LSP1. The data in the bar graphs are presented as the means ± SDs of at least three independent experiments. P values were determined by multiple unpaired t tests ( c ) and paired t tests ( f ). * P < 0.05; ** P < 0.01.

Article Snippet: In some experiments, recombinant HVEM (5 μg/mL; Enzo, #ALX-522-017-C050), PD-1 (5 μg/mL; Enzo, #ENZ-PRT190-0050), BAPTA (5 μM; Invitrogen, #B1205), the PKCβ inhibitor ruboxistaurin (4 μM; MedChemExpress, #HY-10195), procainamide (20 μM; Sigma‒Aldrich, #P9391), hydralazine (20 μM; Sigma‒Aldrich, #H1753), 5-azacytidine (1 μM; Sigma‒Aldrich, #A2385), or ATRA (100 and 1000 nM; Sigma‒Aldrich, #R2625) was added to B cells as indicated.

Techniques: Expressing, Western Blot, Transduction, Control, shRNA, Inhibition

Journal: Oxidative Medicine and Cellular Longevity

Article Title: p66Shc Mediates Mitochondrial Dysfunction Dependent on PKC Activation in Airway Epithelial Cells Induced by Cigarette Smoke

doi: 10.1155/2018/5837123

Figure Lengend Snippet: Pretreatments with PKC β and PKC δ inhibitors ameliorated CSE-induced mitochondrial damage of airway epithelial cells. Mitochondrial reactive oxygen species (ROS) was observed by confocal microscopy (a), and mitochondrial membrane potential (MMP) was determined by flow cytometry (b). Quantitative analysis showed that pretreatments with LY333531 and rottlerin reduced mitochondrial ROS content (c) and increased the levels of MMP (d) and intracellular ATP (e) in Bease-2b cells stimulated with 7.5% CSE for 24 h. All the results are shown as the mean ± SD from three independent experiments. ∗ P < 0.01 versus the control group and # P < 0.05 versus the 7.5% CSE-treated group.

Article Snippet: In addition, cells were also stimulated with 7.5% CSE after the knockdown of p66Shc or 30 min pretreatments with PKC β and PKC δ inhibitors (Sigma).

Techniques: Confocal Microscopy, Flow Cytometry

Journal: Oxidative Medicine and Cellular Longevity

Article Title: p66Shc Mediates Mitochondrial Dysfunction Dependent on PKC Activation in Airway Epithelial Cells Induced by Cigarette Smoke

doi: 10.1155/2018/5837123

Figure Lengend Snippet: Pretreatments with PKC β and PKC δ inhibitors attenuated CSE-induced airway epithelial cell injury. Cell apoptosis was determined by flow cytometry (a), and apoptosis rates of Beas-2b cells treated with 7.5% CSE and PKC β / δ inhibitors were both significantly decreased compared to the cells treated only with 7.5% CSE (b). Pretreatments with LY333531 and rottlerin significantly upregulated cell viability (c) and downregulated culture supernatant concentration of IL-6 and TNF- α (d) in Beas-2b cells exposed to 7.5% CSE. Data are expressed as the mean ± SD from three independent experiments. ∗ P < 0.01 versus the control group and # P < 0.05 versus the 7.5% CSE-treated group.

Article Snippet: In addition, cells were also stimulated with 7.5% CSE after the knockdown of p66Shc or 30 min pretreatments with PKC β and PKC δ inhibitors (Sigma).

Techniques: Flow Cytometry, Concentration Assay