Journal: Cell reports

Article Title: Molecular mechanism of RIPK1 and caspase-8 in homeostatic type I interferon production and regulation

doi: 10.1016/j.celrep.2022.111434

Figure Lengend Snippet:

Article Snippet: H151 , Selleckchem , Cat# S6652.

Techniques: Recombinant, Protease Inhibitor, Western Blot, Reverse Transcription, Cloning, Enzyme-linked Immunosorbent Assay, SYBR Green Assay, Sequencing, Software, Microarray

Journal: bioRxiv

Article Title: “RIG-I mediated neuron-specific IFN type 1 signaling in FUS-ALS induces neurodegeneration and offers new biomarker-driven individualized treatment options for (FUS-)ALS.”

doi: 10.1101/2024.12.02.626340

Figure Lengend Snippet: (A, B) RT-qPCR for a set of ISGs, as indicated on the x-axis in sMNs with a FUS-P525L mutation, normalized to an isogenic control line (A) and three other sMNs lines with distinct C-terminal FUS mutations normalized to two non-isogenic FUS wt sMNs lines (B), unpaired t-test. (C) sketch of the TBK1-IRF3 pathway. TBK1 activation by either RIG-I or cGAS sensing results in downstream transcriptional activation of a type-1 IFN response following the translocation of phosphorylated IRF3 into the nucleus or phosphorylation of p65 at Ser-536. (D) Example western blot scan indicating protein expression of markers of the TBK1-IRF3 pathway in WT and isogenic FUS-P525L sMNs. (E-H) Quantification of (D) normalized to a total protein staining, n=4, unpaired t-test. (I,K) Quantification of RelA and p-RelA WB band intensity, n=3, unpaired t-test. (K) (L) RT-qPCR for selectively IFN-1 dependent ISGs (MX1, IFI44, and STAT1) in FUS-P525L sMNs normalized to isogenic control and (M-O) influence of different treatment strategies on their expression normalized to mock treatment, one-way ANOVA, Tukey’s post hoc. DNA-damaging approaches (etoposide, PARP inhibitor ABT888) did not change their expression in FUS mut or FUS wt . Similarly, the STING inhibitor H151 did not lower the ISG expression in mutant sMNs. Treatment with IFN-beta as a positive control.

Article Snippet: The STING inhibitor H151 was obtained from Tocris (6675) and dissolved in DMSO to a stock of 5 mM.

Techniques: Quantitative RT-PCR, Mutagenesis, Control, Activation Assay, Translocation Assay, Western Blot, Expressing, Staining, Positive Control

Journal: bioRxiv

Article Title: Gram-positive bacteria secrete RNA aptamers to activate human STING for IL-1β release

doi: 10.1101/2021.07.21.453173

Figure Lengend Snippet: (A-D) THP1 (black), CASP1 -/- (blue), ASC -/- (grey), CASP4 -/- (green), cGAS -/- (white) and STING -/- (pattern), MΦs were infected with SA (MOI 10) and GBS (MOI 20), IL-1β production was measured in cell supernetant. (E) THP1, STING -/- , and cGAS -/- MΦs were infected with SA (MOI 10) and GBS (MOI 20), cleaved IL-1β (P17) and caspase-1 (P20) were detected in cell supernetants (Sup) and pro-IL- 1β, pro-caspase-1 and GAPDH in cell lysate by immunoblotting. (F) STING -/- (pattern), STING -/- expressing STING R232 (Black) MΦs were infected with SA (MOI 10) and GBS (MOI 20), IL-1β production was measured in cell supernetant. (G,H) Human blood derived MΦs were pre-treated with increasing concentrations of STING inhibitor (H-151) (100 µM, 50 µM, 10 µM) for 1 h and then infected with SA (MOI 10) and GBS (MOI 20) respectively. IL-1β production was measured in cell supernetant. (I) IL-1β production in wild type THP1 MΦs following transfection with increasing concentrations of SA RNA (RNA+LF) (5 µg/ml, 2.5 µg/ml, 0.1 µg/ml). (J,K,L) THP1 MΦs were pre-treated with increasing concentrations of cytosolic RNase A (RNase A+LF) (100 ng/ml, 50ng/ml, 10 ng/ml) followed by infection with SA. IL-1β production was measured in cell supernetant. Heat inactivated RNase A (100 ng/ml, 50ng/ml, 10 ng/ml) (K) and DNase I (100 ng/ml, 50ng/ml, 10 ng/ml) (L) were used as a control in similar experimental setup. Data shown are mean ±SD (n=3), representative of at least three independent experiments. Asterisks indicate statistically significant differences (∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001).

Article Snippet: Experiments with small molecule inhibitors: THP1 MΦ were pre-treated with MCC950 (5 µM, 10 µM) , Dynasore (150, 100 µM) (Enzo), KCL (60 mM, 45 mM, 75 mM), H-151 (100 µM, 50 µM, 10 µM) (ProbeChem) for 1 h prior to the stimulations.

Techniques: Infection, Western Blot, Expressing, Derivative Assay, Transfection

Journal: Frontiers in Immunology

Article Title: Immunogenic cell death triggered by impaired deubiquitination in multiple myeloma relies on dysregulated type I interferon signaling

doi: 10.3389/fimmu.2023.982720

Figure Lengend Snippet: Analysis of the IFN-stimulated gene (ISG) expression profile in NCI-H929 MM cells exposed to protein homeostasis disruptors. (A) Gene expression of eight typical IFN-stimulated genes ( IFIT1 , IFI27 , IFI44 , IFI44L , ISG15 , MX1 , RSAD2 and SIGLEC1 ) was assayed by RT-qPCR on NCI-H929 MM cell lines after a 12-h exposure to BTZ, ONX0914, RA190, PR619 or DMSO (control), as indicated. Expression levels were normalized to housekeeping genes (RPLP0) and relative quantifications (RQ) are presented as fold change over cells exposed to DMSO. Shown is one representative experiment out of three. (B) Shown are fold change median values of the eight ISG over DMSO measured in three independent experiments. Statistical significance was assessed by paired t test (* p <0.05, *** p <0.001).

Article Snippet: The small-molecule inhibitors RA190, H-151, 4μ8C, C16 and Guanabenz targeting ADRM1/Rpn13, STING, IRE1α, PKR and GADD34, respectively were purchased from Merck Millipore.

Techniques: Expressing, Gene Expression, Quantitative RT-PCR, Control

Journal: Frontiers in Immunology

Article Title: Immunogenic cell death triggered by impaired deubiquitination in multiple myeloma relies on dysregulated type I interferon signaling

doi: 10.3389/fimmu.2023.982720

Figure Lengend Snippet: Western-blot analysis of the ubiquitin, ISR and UPR expression profiles in NCI-H929 cells exposed to BTZ, ONX-0914, RA190 or PR619. (A) NCI-H929 exposed to PR619 (1,5 µM) or left untreated were subjected to protein extraction and subsequent SDS-PAGE/western blotting using antibodies specific for ubiquitin and actin (loading control), as indicated. Shown is one representative experiment out of three. (B) Equal amounts of protein lysates derived from NCI-H929 exposed to a 12-h treatment with DMSO, BTZ (50 nM), ONX-0914 (50 nM), RA190 (50 nM) or PR619 (1,5 µM) were analyzed by SDS-PAGE/western-blotting using antibodies directed against PKR, (p)PKR, GCN2, (p)GCN2, eIF2α, (p)eIF2α, 4E-BP1, (p)4E-BP1 and tubulin (loading control), as indicated. Shown is one representative experiment out of three. (C) NCI-H929 whole cell-lysates described in (B) were further assessed for their contents in PERK, (p)PERK, IRE1, (p)IRE1, ATF6 by SDS-PAGE/western-blotting, as indicated. Equal protein loading was ensured by probing the membranes with an actin antibody. Shown is one representative experiment out of three. (D) Densitometric analysis of phosphorylated of PERK, IRE1, PKR and GCN2 normalized to total proteins and reported as foldchange relative to DMSO.

Article Snippet: The small-molecule inhibitors RA190, H-151, 4μ8C, C16 and Guanabenz targeting ADRM1/Rpn13, STING, IRE1α, PKR and GADD34, respectively were purchased from Merck Millipore.

Techniques: Western Blot, Ubiquitin Proteomics, Expressing, Protein Extraction, SDS Page, Control, Derivative Assay

Journal: Frontiers in Immunology

Article Title: Immunogenic cell death triggered by impaired deubiquitination in multiple myeloma relies on dysregulated type I interferon signaling

doi: 10.3389/fimmu.2023.982720

Figure Lengend Snippet: Effects of ONX0914- and RA190-induced cell death on the ability of NCI-H929 cells to deliver stimulatory signals to DC. (A) Histogram overlays of flow cytometry analysis of DC cell surface expression of CD80, CD83 and CD86 upon a 24 h-incubation with NCI-H929 dead cells obtained from treatments with ONX0914 (red line), RA190 (brown line), BTZ (purple line) or PR619 (green line), as indicated. Negative control in this experiment consisted of unloaded day 5-immature DC (blue line). Shown is one representative experiment out of three. (B) Measurements of the percentage of DC positive for CD80, CD83 or CD86 following co-culture with ONX0914-, RA190-, BTZ- or PR619-induced NCI-H929 apoptotic cells, as indicated. Shown is the median from three independent experiments. Statistical significance was assessed by paired t test where *indicates p <0.05 and *** indicates p <0.001, ns, not significant.

Article Snippet: The small-molecule inhibitors RA190, H-151, 4μ8C, C16 and Guanabenz targeting ADRM1/Rpn13, STING, IRE1α, PKR and GADD34, respectively were purchased from Merck Millipore.

Techniques: Flow Cytometry, Expressing, Incubation, Negative Control, Co-Culture Assay

Journal: Frontiers in Immunology

Article Title: Immunogenic cell death triggered by impaired deubiquitination in multiple myeloma relies on dysregulated type I interferon signaling

doi: 10.3389/fimmu.2023.982720

Figure Lengend Snippet: Measurements of calreticulin (CRL) cell surface expression and ATP extracellular release by NCI-H929 cells treated with BTZ, ONX0914, RA190 or PR619. (A) Flow cytometry histogram overlays of CRL cell surface expression by NCI-H929 cells following a 24 or 48h-treatment with DMSO (red line), BTZ (blue line), ONX0914 (brown line), RA190 (purple line) or PR619 (green line), as indicated. Shown is one representative experiment out of three. (B) Measurements of the percentage of NCI-H929 cells positive for CRL after a 24 or 48 h-incubation with DMSO, BTZ, ONX0914, RA190 or PR619, as indicated. Shown is the median calculated from three independent experiments. Statistical significance was assessed by paired t test where *indicates p <0.05 and ** indicates p <0.01. (C) Bioluminescence analysis of extracellular ATP levels in supernatants from NCI-H929 cells subjected to a 6 or 24 h-treatment with DMSO, BTZ, ONX0914, RA190 or PR619. Shown is the median of the relative light units (RLU) measured following a 5 min-incubation with the luciferase-containing assay medium and calculated from three independent experiments. Statistical significance was assessed by paired t test where * indicates p <0.05.

Article Snippet: The small-molecule inhibitors RA190, H-151, 4μ8C, C16 and Guanabenz targeting ADRM1/Rpn13, STING, IRE1α, PKR and GADD34, respectively were purchased from Merck Millipore.

Techniques: Expressing, Flow Cytometry, Incubation, Luciferase

Journal: F1000Research

Article Title: Determinants of drug entry into the developing brain

doi: 10.12688/f1000research.20078.1

Figure Lengend Snippet: Numbers of animals (n) used in the brain and CSF drug permeability experiments. The n numbers listed in brackets are where the numbers of CSF measurements were different from the number of brain measurements. No animal died before the termination of an experiment. E19 and P4 pups were littermates and included both sexes. The differences in pup numbers between groups is due to natural variation in litter sizes. P4 and adult experiments were terminated 30 minutes after tracer administration, whereas the E19 embryo experiments were terminated between 30 minutes and 2.5 hours post-injection due to the time required to sequentially sample each embryo. Acute: experiments involved a single injection of unlabelled drug mixed with a tracer amount of radiolabelled drug. Chronic: animals received multiple drug injections of unlabelled drug over a period of five days (see Methods) with the final injection including the radiolabelled tracer. Adults were non-pregnant females.

Article Snippet: Paracetamol , [2,6- 3 H] , 151 , American Radiolabeled Chemicals, Inc. , ART 0679.

Techniques: Permeability, Injection

Journal: F1000Research

Article Title: Determinants of drug entry into the developing brain

doi: 10.12688/f1000research.20078.1

Figure Lengend Snippet: List of radio-labelled markers, their suppliers and product codes.

Article Snippet: Paracetamol , [2,6- 3 H] , 151 , American Radiolabeled Chemicals, Inc. , ART 0679.

Techniques: Molecular Weight

Journal: F1000Research

Article Title: Determinants of drug entry into the developing brain

doi: 10.12688/f1000research.20078.1

Figure Lengend Snippet: Bars are group means with individual data points shown, * p<0.05, ** p<0.01, *** P<0.001. At E19, paracetamol was administered by i.p. injection to the mother. Individual fetuses were serially sampled starting at 30 minutes following maternal injection up to approximately 2.5 hours (see for times of sampling and maternal and fetal plasma paracetamol levels). Adult and P4 animals were injected i.p. and samples taken at 30 minutes. Note that for both acute and chronic treatment groups, the ratios were higher in brain and CSF in younger animals. At E19, following chronic doses, ratios for both brain and CSF were higher; in the adults they were lower.

Article Snippet: Paracetamol , [2,6- 3 H] , 151 , American Radiolabeled Chemicals, Inc. , ART 0679.

Techniques: Injection, Sampling, Clinical Proteomics

Journal: F1000Research

Article Title: Determinants of drug entry into the developing brain

doi: 10.12688/f1000research.20078.1

Figure Lengend Snippet: Expression of ABC transporters in brain cortex, choroid plexus (lateral ventricular) and placenta (E19) in E19, P4 and adult rats, RT-qPCR. The results are fold change differences between the chronic and acute treatments. They confirm the expression of MRP1-5 ( abcc1-5 ), BCRP (abcg2) and P-glycoprotein/PGP ( abcb1a, abcb1b ) in these rat tissues. Statistically significant differences in transcript numbers in the chronic treatment group (p <0.05) are indicated by ↑ where there was up-regulated expression of the target and by ↓ where there was down-regulated expression. * indicates a large fold change increase that did not reach statistical significance due to extremely low expression in all acute and chronically treated animals except for two animals that expressed the transporter at a high level. Note the near total absence of changes in expression in brain and choroid plexus at E19 compared to the large number of changes at P4.

Article Snippet: Paracetamol , [2,6- 3 H] , 151 , American Radiolabeled Chemicals, Inc. , ART 0679.

Techniques: Expressing

Journal: F1000Research

Article Title: Determinants of drug entry into the developing brain

doi: 10.12688/f1000research.20078.1

Figure Lengend Snippet: ( A ) digoxin, ( B ) cimetidine and ( C ) paracetamol in E19 fetal (green triangles), P4 postnatal (blue squares) and adult (red circles) rats plotted against their lipid solubility (LogD Octanol partition coefficient) and compared with the “passive” permeability markers sucrose, L-glucose and glycerol. Filled symbols indicate acute experiments (30 minutes after IP injection) and open symbols indicate chronic experiments (after twice daily IP injections over five days). See and for full data and n numbers. Ratios less than 100% indicate restricted entry of the drug or marker into brain. Digoxin, despite being the most lipid soluble of the drugs was the most restricted at all ages in both the acute and chronic treatments. Cimetidine was similarly restricted at P4 and in adults, but less so at E19. Paracetamol was the least restricted of the drugs. Also note that following chronic treatment, paracetamol entry into brain decreased in adults, but was increased at E19 (direction of significant changes indicated by arrows). Chronic treatment also reduced the entry of digoxin into adult brain adults, but not at P4 and E19.

Article Snippet: Paracetamol , [2,6- 3 H] , 151 , American Radiolabeled Chemicals, Inc. , ART 0679.

Techniques: Solubility, Permeability, Injection, Marker

Journal: F1000Research

Article Title: Determinants of drug entry into the developing brain

doi: 10.12688/f1000research.20078.1

Figure Lengend Snippet: ( A ) digoxin, ( B ) cimetidine and ( C ) paracetamol in E19 fetal (green triangles), P4 postnatal (blue squares) and adult (red circles) rats plotted against their lipid solubility (LogD Octanol partition coefficient) and compared with the “passive” permeability markers sucrose, L-glucose and glycerol. Filled symbols indicate acute experiments (30 minutes after IP injection) and open symbols indicate chronic experiments (after twice daily IP injections over five days). See and for full data and n numbers. Ratios less than 100% indicate restricted entry of the drug or marker into CSF. Digoxin, despite being the most lipid soluble of the drugs, was the most restricted at all ages in both the acute and chronic treatments. Cimetidine was similarly restricted at P4 and in adults, but less so at E19. Paracetamol was the least restricted of the drugs. Also note that following chronic treatment, paracetamol entry into CSF decreased in adults, but was increased at E19 (direction of significant changes indicated by arrows). Chronic treatment did not significantly affect the entry of cimetidine into CSF at any of the three ages investigated.

Article Snippet: Paracetamol , [2,6- 3 H] , 151 , American Radiolabeled Chemicals, Inc. , ART 0679.

Techniques: Solubility, Permeability, Injection, Marker

Journal: F1000Research

Article Title: Determinants of drug entry into the developing brain

doi: 10.12688/f1000research.20078.1

Figure Lengend Snippet: Note that the maternal plasma levels for both acute and chronic experiments declined progressively for all three drugs throughout the 2–2.5 hours experimental period, but the fetal plasma levels were stable for digoxin and cimetidine during the same period. The paracetamol levels in fetal plasma declined during this period. For digoxin and cimetidine, the levels of radiolabelled drugs in acute and chronic experiments were similar, but for paracetamol the levels in maternal and fetal plasma were much higher with chronic treatment. The much lower levels for each drug in fetal plasma indicates a substantial restriction of drug transfer across the placenta. Lines fitted by Least Squares Linear Regression, curve fitted by Least Squares Exponential Decay (one phase).

Article Snippet: Paracetamol , [2,6- 3 H] , 151 , American Radiolabeled Chemicals, Inc. , ART 0679.

Techniques: Clinical Proteomics

Journal: F1000Research

Article Title: Determinants of drug entry into the developing brain

doi: 10.12688/f1000research.20078.1

Figure Lengend Snippet: [ 3 H]-labelled “passive” permeability markers L-glucose and glycerol (both open circles) are shown; along with [ 3 H]-labelled drugs digoxin (blue triangles), cimetidine (purple diamond) and paracetamol (pink hexagon). Filled symbols indicate acute experiments and open symbols indicate chronic experiments. Values are means, see for full data and and for n numbers. Note that all three drugs showed a degree of restriction (values below the trend of passive permeability) provided by the placental barrier. There was no difference between acute and chronic treatment groups for any of the drugs tested.

Article Snippet: Paracetamol , [2,6- 3 H] , 151 , American Radiolabeled Chemicals, Inc. , ART 0679.

Techniques: Permeability