Journal: bioRxiv

Article Title: BLOC1S1 control of vacuolar organelle fidelity modulates T H 2 cell immunity and allergy susceptibility

doi: 10.1101/2024.03.21.586144

Figure Lengend Snippet: (A) Representative immunoblots for STING, P-IκBα, IκBα, P-NF-κB p65, NF-κB p65, GATA3, P- STAT6, STAT6 and β-actin from CTRL and TKO CD4 + T cells following DMSO or H151 (500 nM) incubation for 48 hours. (B) Protein quantitation and ratio of STING/β-actin, P-IKBα/IKBα, P-NF-kB P65/NF-kB P65, GATA3/β-actin and P-STAT6/STAT6 by densitometry analysis (n=6-7 per group). (C) IL-4, IL-5 and IL-13 cytokine release in activated CD4 + T cells treated with either DMSO or H151 (500nM) (n=6-7 per group). (D) Representative immunoblots for STING, P-IκBα, IκBα, P-NF-κB p65, NF-kB p65, GATA3 and β-actin from CTRL and TKO CD4 + T cells incubated with negative control (N.C.) or with STING siRNA. (E) Protein quantitation and ratio of STING/β-actin, P-IκBα/IκBα, P-NF-κB P65/NF-κB P65 and GATA3/β-actin by densitometry analysis (n=6 per group). (F) IL-4, IL-5 and IL-13 cytokine release in activated CD4 + T cells incubated with either N.C. or STING siRNA (n=9-18 per group). (G) Schematic representation of proposed mechanistic pathway. Values represent mean ± SEM. *p<0.05, **p<0.01, ***p<0.001 vs control mice by two-way ANOVA followed by the Tukey’s post hoc test or unpaired two-tailed student-t-test.

Article Snippet: The following inhibitors were added to the cells for 24 hours before harvesting: the NF-κB inhibitor JSH23 (2 μM, Tocris Bioscience), STING inhibitor H151 (5 μM, Tocris Bioscience), or Rapamycin (2 μM, Selleckchem).

Techniques: Western Blot, Incubation, Protein Quantitation, Negative Control, Control, Two Tailed Test

Journal: eLife

Article Title: Ephrin-B3 controls excitatory synapse density through cell-cell competition for EphBs

doi: 10.7554/eLife.41563

Figure Lengend Snippet:

Article Snippet: Antibody , Goat polyclonal anti-mouse ATTO 425 , Rockland antibodies , Cat. # 611-151-122, RRID: AB_10893217 , (1:250).

Techniques: Knock-Out, Recombinant, Plasmid Preparation, shRNA, Multiplex Assay, Software

Journal: Nature Communications

Article Title: Loss of Zfp335 triggers cGAS/STING-dependent apoptosis of post-β selection thymocytes

doi: 10.1038/s41467-022-33610-4

Figure Lengend Snippet: a Schematic diagram of inhibitors (RU.521 or H-151) or agonists (CMA) used to study cGAS/STING-dependent apoptosis of DN4 thymocytes. Representative histograms ( b ) and quantification ( c ) of Annexin V binding for WT ( n = 5 mice) or Zfp335cKO ( n = 4 mice) DN4 thymoctes treated with cGAS (RU.521) or STING (H-151) inhibitors or vehicle control and cultured on OP9-DL1 stromal cells for 3 days. Total thymocyte ( d ), DN, DP, CD4SP, and CD8SP or DN1-DN4 cell numbers ( e , f ) or frequencies ( g , h ) for Zfp335cKO mice treated with H-151 ( n = 8 mice) or vehicle ( n = 6 mice) in vivo for 7 days. i Quantification of Annexin V binding among DN4 cells from R26 LSL-Cas9 Tcrd CreERT2 thymocytes transduced with gRNA-expressing retroviruses and cultured for three days on OP9-DL1 cells with 4-hydroxytamoxifen. n = 3 biological replicates pooled from 3 mice each for each dual gRNA transduction. Percent apoptosis induced by small molecule activation of STING among WT thymocyte subsets treated with 25 µg/mL ( j ) or 250 µg/mL CMA ( k ), and Zfp335cKO Bcl2-Tg ( l ), or αCD3-treated Rag2 −/− thymocyte subsets ( m ) treated with 25 µg/mL CMA. Panels j and k were from the same experiments. n = 6 mice ( j , k ), n = 8 mice ( l ), or n = 7 mice ( m ). P -values determined by two-tailed Mann–Whitney U-test ( d ) or two-way ANOVA with post hoc Tukey’s test ( c ) or Sidak’s test ( e – h , j – m ) or one-way ANOVA with post hoc Tukey’s test ( i ). Data shown are compiled from two ( b , c , m ), three ( i – l ), or five ( d – h ) independent experiments utilizing male and female mice. Plots show mean ± SEM. Source data are provided as a Source Data file.

Article Snippet: Mice were administered 750 pmol (210 µg) of H-151 (Cayman Chemicals) or vehicle via intraperitoneal injection daily for 7 days beginning at 7 weeks of age.

Techniques: Binding Assay, Cell Culture, In Vivo, Transduction, Expressing, Activation Assay, Two Tailed Test, MANN-WHITNEY

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