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:

Article Title: p300 and estrogen receptor cooperatively activate transcription via differential enhancement of initiation and reinitiation

doi:

Figure Lengend Snippet: Purification of human ER and L540Q variant. (A) Schematic diagram of wild-type and epitope-tagged ERs. The locations of the amino-terminal Flag epitope tag and the Leu-540 → Gln (L540Q) point mutation are indicated. (B) Synthesis and purification of epitope-tagged ERs. Flag-tagged ER and ER(L540Q) were synthesized in Sf9 cells by using a baculovirus expression vector and affinity-purified with monoclonal antibodies that recognize the FLAG epitope. The proteins were subjected to 8% polyacrylamide–SDS gel electrophoresis. (Left) Total protein was visualized by staining with Coomassie Brilliant Blue R-250; (right) ERs were detected by Western blot analysis with anti-ER antibodies. (C) Ligand-binding activity of purified recombinant ERs. The 3H–E2 binding activity of approximately equimolar amounts of purified ERs was determined by ligand binding assays. Specific binding activity was determined by analyzing a second set of samples in the presence of a 200-fold excess of unlabeled E2. The net binding of 3H–E2 (in fmoles) is shown. (D) DNA-binding activity of purified recombinant ERs. Gel mobility shift assays were performed with approximately equimolar amounts of purified ERs. The samples were incubated with a 32P-end-labeled double-stranded oligonucleotide containing a consensus ERE sequence in the presence or the absence of E2 and then analyzed with a nondenaturing 4.8% polyacrylamide gel.

Article Snippet: Western blot assays were carried out with a monoclonal antibody (H-151; Stressgen) directed against the carboxyl-terminus of ER or with a polyclonal antiserum raised against a bacterially expressed, affinity-purified, His 6 -tagged amino-terminal fragment (amino acids 1–113) of ER.

Techniques: Purification, Variant Assay, FLAG-tag, Mutagenesis, Synthesized, Expressing, Plasmid Preparation, Affinity Purification, Bioprocessing, SDS-Gel, Electrophoresis, Staining, Western Blot, Ligand Binding Assay, Activity Assay, Recombinant, Binding Assay, Mobility Shift, Incubation, Labeling, Sequencing

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