Journal: bioRxiv

Article Title: RAS activation via CRLF2 signaling is a widespread mechanism in Down syndrome acute lymphoblastic leukemia regardless of RAS mutations

doi: 10.1101/2020.02.03.931725

Figure Lengend Snippet: Western blot analysis of the murine pro B cell line Ba/F3. Cells were stably transfected with human JAK2 R683G and/or human CRLF2 (see Supplementary-Fig.S1C and Supplementary-Fig.S1D) and cultured in IL-3-containing medium. All cells were then starved from IL-3 and cells were lysed. Each cell lysate was split up for analysis in RAS-GTP pull-down assay and for total proteins. An SDS-PAGE followed by Western blotting was performed. (A) Left-hand side blot shows the RAS-GTP (activated RAS) pull-down while the right-hand side blots show whole cell lysates of the same samples. Antibody-targets are labeled on the right side of each image with black arrows marking the respective protein band; the antibody against HA-tag shows the expression of the human JAK2 construct. The experiment was repeated 4 times independently. (B) Quantification of (A) for active RAS (RAS-GTP) normalized to its level in untransfected cells. Error bars are SD and P -values were determined in one-way ANOVA and post-hoc Bonferroni multiple comparison.

Article Snippet: The primary antibodies against β-actin (Cat.#ab8227; Western blot (WB)1:10,000) and GRB2 (Cat.#ab86713; PLA 1:100) were purchased from Abcam (Cambridge, UK); the antibody against CRLF2 (Cat.#AF981; WB 1 μg/mL) was purchased from R&D Systems (Minneapolis, US).

Techniques: Western Blot, Stable Transfection, Transfection, Cell Culture, Pull Down Assay, SDS Page, Labeling, Expressing, Construct, Comparison

Journal: bioRxiv

Article Title: RAS activation via CRLF2 signaling is a widespread mechanism in Down syndrome acute lymphoblastic leukemia regardless of RAS mutations

doi: 10.1101/2020.02.03.931725

Figure Lengend Snippet: MUTZ-5 cells (Human Ph-like B-ALL cells bearing CRLF2-rearranged and spontaneous JAK2R683G mutation) were stimulated with 20 ng/mL human TSLP (maximal effective TSLP-concentration, Supplementary-Fig.S3B) for 10 min before cell lysis. Each cell lysate was split up for analysis in RAS-GTP pull-down assay and for total protein signal. (A) RAS-GTP pull-down and lysate samples were loaded on separate gels. An SDS-PAGE followed by Western blotting was performed. To assess the total protein and phosphorylated protein amounts on the same PVDF-membrane, each membrane part was stripped and reprobed with new antibodies. RAS-GTP pull-down samples are on the left side while the right-hand side blots show whole cell lysates of the same samples. Antibody-targets are labeled on the right side of each image with black arrows indicating the respective protein band. The grey arrow shows the unspecific signal of the GST-RAS binding domain (RBD) used in the active RAS pull-down assay acting as a loading control. The experiment was repeated 5 times independently and the graphs show the quantification for active RAS (RAS-GTP), phosphorylated MEK1/2 (phospho-MEK1/2), JAK2 (phospho-JAK2), and PTPN11 (phosho-PTPN11). Beta-actin and total protein signals were used as a loading control to normalize samples. (B) A blot separate from (A) demonstrates the TSLP-inducibility of RAS-effector bRAF. (C) RAS-GTP quantification of 5 independent ELISA experiments in which RAS activity of TSLP-induced MUTZ-5 cells was measured using a different, ELISA-specific active RAS pull-down assay. (D) TSLP-induced MUTZ-5 cells were probed for the presence of activated isoforms KRAS-GTP, HRAS-GTP, or NRAS-GTP (blots on the left). The blots on the right show the total expression of the respective RAS proteins and the graphs show the signal fold change over uninduced MUTZ-5 cells for KRAS-GTP, HRAS-GTP and NRAS-GTP of 4 independent experiments. All error bars are SD; P -values were calculated using Student’s T-test and are adjusted with a Bonferroni-correction for sequential multiple comparison.

Article Snippet: The primary antibodies against β-actin (Cat.#ab8227; Western blot (WB)1:10,000) and GRB2 (Cat.#ab86713; PLA 1:100) were purchased from Abcam (Cambridge, UK); the antibody against CRLF2 (Cat.#AF981; WB 1 μg/mL) was purchased from R&D Systems (Minneapolis, US).

Techniques: Mutagenesis, Concentration Assay, Lysis, Pull Down Assay, SDS Page, Western Blot, Membrane, Labeling, Binding Assay, Control, Enzyme-linked Immunosorbent Assay, Activity Assay, Expressing, Comparison

Journal: bioRxiv

Article Title: RAS activation via CRLF2 signaling is a widespread mechanism in Down syndrome acute lymphoblastic leukemia regardless of RAS mutations

doi: 10.1101/2020.02.03.931725

Figure Lengend Snippet: Primary presentation samples of DS-ALL patients were cultured for 2 days (see Supplementary-Fig.S4A legend for details) and then induced for 10 min with 20 ng/mL TSLP (or not induced) in serum-reduced medium. Lysates were analyzed for RAS activity in WB pull-down (A) or ELISA (B) using an ELISA-specific RAS pull-down assay. (A) Each lysate was split up for analysis in Western blot RAS-GTP pull-down assay and for total protein signal. RAS-GTP pull-down (left) and lysate samples (right) were loaded on separate gels. An SDS-PAGE followed by Western blotting was performed. To assess the total protein and phosphorylated protein amounts on the same PVDF-membrane, membranes were stripped and reprobed with new antibodies. Antibody-targets are labeled on the right side of each image with black arrows indicating the respective protein band; the grey arrow shows the loading of the GST-RBD in the pull-down assay. (B) The RAS activity pattern in the patient samples from (A) was confirmed via ELISA measurement of RAS-activity in aliquots that were independently thawed and processed as described above The line graph illustrates the four main patterns observed for RAS activity in primary ALL patient samples. (C) Shows an overview of the ELISA-measured RAS activity for the DS-ALL cohort at diagnosis (not enough cell material was available for DS26, DS29 and DS30). The RAS-GTP pull-down for ELISA was performed on lysates from cells at minimum 75% viability at a 100 ng/μL total protein concentration. In parallel, uninduced MUTZ-5 cells were subjected to the same treatment as the primary patient cells and were used to normalize all RAS activities. Brackets on top indicate the groups of the four RAS activity patterns presented in (A, B). For visualization purposes only in this graph, basal RAS activity over 50% of MUTZ-5 basal RAS activity was grouped as high RAS activity while an increase by at least 10% RAS-GTP in TSLP-stimulated samples over uninduced samples in ELISA was classed as TSLP-inducible RAS. For visualization purposes only in this graph, phosphorylation levels measured in WB for JAK2 were categorized as –(negative) = 0.00-0.05; + = 0.05-0.50; ++ = 0.50-1.00; +++ =1.00-2.00, and CRLF2 protein levels were categorized as –(negative) = 0.00-0.05; + = 0.05-0.20; ++ = 0.20-0.50; +++ =0.50-1.50. None of the arbitrary threshold groupings defined above were used in any of the PCA or clustering analysis shown later ( , Supplementary-FigS5). Known CRLF2-rearrangements are marked (R). All values are normalized to those measured for uninduced MUTZ-5 cells.). Outcome of leukemia is given (white = good outcome, black = poor outcome), and the presence of RAS mutations (blue) or JAK2 mutations (red) are specified (grey means unsequenced samples). The groups at the right end of the bar graph (separated by the black bar) show average RAS activities for patient/sample groups other than DS-ALL-diagnosis: Non-DS (NDS) at presentation, DS complete remission (CR) and DS/NDS at relapse. For an overview of the complete Western blot data and the quantified activities and protein expression of STAT5, JAK2, MEK1/2, ERK1/2 and rpS6 of all individual samples, see Supplementary-Fig.S4.

Article Snippet: The primary antibodies against β-actin (Cat.#ab8227; Western blot (WB)1:10,000) and GRB2 (Cat.#ab86713; PLA 1:100) were purchased from Abcam (Cambridge, UK); the antibody against CRLF2 (Cat.#AF981; WB 1 μg/mL) was purchased from R&D Systems (Minneapolis, US).

Techniques: Cell Culture, Activity Assay, Enzyme-linked Immunosorbent Assay, Pull Down Assay, Western Blot, SDS Page, Membrane, Labeling, Biomarker Discovery, Protein Concentration, Phospho-proteomics, Expressing

Journal: bioRxiv

Article Title: RAS activation via CRLF2 signaling is a widespread mechanism in Down syndrome acute lymphoblastic leukemia regardless of RAS mutations

doi: 10.1101/2020.02.03.931725

Figure Lengend Snippet: (A) A principal component analysis (PCA) was performed on the quantified data of (data was given as continuous variables; no cutoffs or pre-grouping were used) for the DS-ALL cohort at diagnosis, and (where available) at remission and relapse, as well as primary presentation and relapse samples from Non-DS ALL patients. Top view of the PCA mapping for all 6 measured protein activities (basal and TSLP-induced) as well as CRLF2 protein expression of all analyzed samples along the calculated principle components (for PCA calculated results see Supplementary-Fig.S5A). K -means clustering (with k set to 4 to achieve minimal class-class deviation, Supplementary-Fig.S5B) grouped samples into clusters 1 to 4 (listed in Supplementary-Fig.S5C). Sample data points are colored according to the unsupervised clustering. (B) PCA Clusters 1 and 2 contain all samples of the DS-ALL diagnosis cohort and were analyzed according to their outcome: A Fisher’s exact test determined the P -value between the number of good and poor outcomes between the two clusters (bar graph). (C) Kaplan–Meier curves of cluster 1 (SR = standard risk) and cluster 2 (HR = high risk) DS-ALL patients. Table show a Cox proportional-hazards model for protein activity score (PCA-derived principle component from all quantified protein activities at basal and TSLP-induced level) together with CRLF2-protein expression level (for CRLF2+ samples), NCI risk groups (SR: age at diagnosis 1-10 yr and WBC < 50.000/µL; HR = children age > 10 yr and/or WBC > 50.000/µL; or unknown), and presence of activating JAK2 mutations. Reverse Kaplan-Meier median follow-up for N=20 DS-ALL was 18.4 years. Patient numbers at risk for each year are given in the table below the survival curve. (D) The means of all analyzed basal or TSLP-induced protein activities are compared between the SR group (DS-ALL patients in PCA cluster 1) and the HR group (DS-ALL patients in PCA cluster 2). All error bars are SD; P -values were calculated using Student’s T-test and are adjusted with a Bonferroni-correction for sequential multiple comparison. For visibility, significant P -values were additionally highlighted with * ( P < .05) or ** ( P < .01).

Article Snippet: The primary antibodies against β-actin (Cat.#ab8227; Western blot (WB)1:10,000) and GRB2 (Cat.#ab86713; PLA 1:100) were purchased from Abcam (Cambridge, UK); the antibody against CRLF2 (Cat.#AF981; WB 1 μg/mL) was purchased from R&D Systems (Minneapolis, US).

Techniques: Biomarker Discovery, Expressing, Activity Assay, Derivative Assay, Comparison

Journal: PLoS Pathogens

Article Title: Synergistic Induction of Interferon α through TLR-3 and TLR-9 Agonists Identifies CD21 as Interferon α Receptor for the B Cell Response

doi: 10.1371/journal.ppat.1003233

Figure Lengend Snippet: (A) Spleen cells from cotton rats were stimulated with Concanavalin A for 72 hours and lymphoblasts purified by centrifugation on a ficoll-gradient. Subsequently, cells were stained with antisera specific for the human interferon receptor to test for crossreactive binding to cotton rat cells and analyzed by flow cytometry. Antibody specific for human interferon alpha receptor-1 (IFNAR-1) did not react with cotton rat cells (left) whereas antibody specific for human interferon alpha receptor-2 (IFNAR-2) did (right). IFNR specific antibody is shown as black line, controls as gray areas. B. IFNAR-2 antibody was added to cotton rat spleen cells. One hour later different concentrations of a combination of ODN2216 and poly I:C was added. 24 hours later supernatant was harvested and tested for the presence of type I interferon. The data is combined from two independent experiments. Significant differences were seen between cells treated with the combination of ODN2216 and poly I:C alone, or the combination of ODN2216 and poly I:C with antibody. IFNAR-1 antibody (not reactive with cotton rat cells) had no effect. C. Cotton rat spleen cells were stimulated with ODN 2216 or poly I:C, with or without the addition of recombinant cotton rat interferon alpha. (One unit equals one picogram of type I interferon.) 24 hours later supernatant was harvested and tested for the presence of type I interferon. The data is combined from two independent experiments, and significance was compared to cells without type I interferon treatment. Statistical analysis was done by ANOVA (* p<0.05, ** p<0.01, *** p<0.001).

Article Snippet: Neutralizing goat antibodies against cotton rat interferon alpha and IL-6 were purchased from R&D systems.

Techniques: Purification, Centrifugation, Staining, Binding Assay, Flow Cytometry, Recombinant

Journal: PLoS Pathogens

Article Title: Synergistic Induction of Interferon α through TLR-3 and TLR-9 Agonists Identifies CD21 as Interferon α Receptor for the B Cell Response

doi: 10.1371/journal.ppat.1003233

Figure Lengend Snippet: A) The number of MeV-specific B cells was measured from bone marrow cells of MeV-immune cotton rats. The addition of ODN 2216 and poly I:C individually and in combination increased B cell numbers whereas the addition of sera neutralizing cotton rat interferon alpha and IL-6 reduced this stimulation. Each bar graph represents the mean ± SD of triplicate wells. B) Cotton rats were immunized intranasally (upper panel) or subcutaneously (lower panel) with MeV, or MeV with ODN 2216 and/or pI:C in the presence or absence of human MeV-specific IgG (neutralization titer of 100) which had been injected intraperitoneally one day before immunization. Sera were collected at seven weeks post vaccination and the titer of neutralizing antibody was determined by neutralization assay. Each bar graph represents the average titer of four animals ± SD. The experiment is representative of three experiments. Statistical analysis was done by ANOVA (* p<0.05, ** p<0.01, *** p<0.001).

Article Snippet: Neutralizing goat antibodies against cotton rat interferon alpha and IL-6 were purchased from R&D systems.

Techniques: Neutralization, Injection

Journal: Frontiers in Immunology

Article Title: Critical Role for the NLRP3 Inflammasome in Mediating IL-1β Production in Shigella sonnei -Infected Macrophages

doi: 10.3389/fimmu.2020.01115

Figure Lengend Snippet: S. sonnei induces the secretion of IL-1β, IL-18, NLRP3, ASC, and active caspase-1 in macrophages. (A) J774A.1 macrophages, THP-1 macrophages, PBMCs or BMDM were primed with 1 μg/ml LPS for 4 h and then infected with S. sonnei for an additional 20 h. The levels of IL-1β in the supernatants were measured by ELISA. (B–E) J774A.1 macrophages or THP-1 macrophages were primed with 1 μg/ml LPS for 4 h followed and then infected with S. sonnei for an additional 20 h or stimulated with 5 mM ATP for an additional 0.5 h. The levels of IL-1β (B) , IL-18 (C) , caspase-1 (D) , NLRP3, and ASC (E) in the supernatants were measured by Western blotting. The ELISA data are expressed as the mean ± SD of four separate experiments. The Western blotting results are representative of three different experiments. * and *** indicate significant differences at the levels of p < 0.05 and p < 0.001, respectively, compared to untreated control cells (one-way ANOVA with Dunnett's multiple comparisons test).

Article Snippet: Antibodies against IL-1β (AB-401-NA, polyclonal antibody) were purchased from R&D Systems (Minneapolis, MN).

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Western Blot, Control

Journal: Frontiers in Immunology

Article Title: Critical Role for the NLRP3 Inflammasome in Mediating IL-1β Production in Shigella sonnei -Infected Macrophages

doi: 10.3389/fimmu.2020.01115

Figure Lengend Snippet: S. sonnei induces IL-1β secretion through the NLRP3 inflammasome. (A,B) J774A.1 macrophages or BMDM were primed with 1 μg/ml LPS for 4 h and then treated with YVAD-CHO (A) or MCC950 (B) for 0.5 h. The cells were then infected with 50 MOI S. sonnei for an additional 20 h. The levels of IL-1β in the supernatants were measured by ELISA. (C,D) Wild-type or NLRP3 knockout J774A.1 macrophages were primed with 1 μg/ml LPS for 4 h and then infected with 50 MOI S. sonnei for an additional 20 h. The levels of IL-1β (C) and caspase-1 (D) in the supernatants were measured by ELISA and Western blotting, respectively. The ELISA data are expressed as the mean ± SD of four separate experiments. The Western blotting results are representative of three different experiments. * and *** indicate significant differences at the levels of p < 0.05 and p < 0.001, respectively, compared to S. sonnei -infected cells (A,B) or untreated control cells (C) (one-way ANOVA with Dunnett's multiple comparisons test).

Article Snippet: Antibodies against IL-1β (AB-401-NA, polyclonal antibody) were purchased from R&D Systems (Minneapolis, MN).

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Knock-Out, Western Blot, Control

Journal: Frontiers in Immunology

Article Title: Critical Role for the NLRP3 Inflammasome in Mediating IL-1β Production in Shigella sonnei -Infected Macrophages

doi: 10.3389/fimmu.2020.01115

Figure Lengend Snippet: S. sonnei activates the NLRP3 inflammasome through P 2 X 7 receptor-mediated potassium efflux. (A–C) J774A.1 macrophages were primed with 1 μg/ml LPS for 4 h and then treated with KCl (A) , glibenclamide (B) , probenecid or carbenoxolone (C) for 0.5 h. The cells were then infected with 50 MOI S. sonnei for an additional 20 h. The levels of IL-1β and caspase-1 in the supernatants were measured by ELISA and Western blotting, respectively. (D,E) Mock or P 2 X 7 knockdown J774A.1 macrophages were primed with 1 μg/ml LPS for 4 h and then infected with 50 MOI S. sonnei for an additional 20 h or stimulated with 5 mM ATP for an additional 0.5 h. The levels of IL-1β (D) and caspase-1 (E) in the supernatants were measured by ELISA and Western blotting, respectively. The levels of P 2 X 7 in mock or P 2 X 7 knockdown J774A.1 macrophages were measured by Western blotting and flow cytometry (D) . (F) Mock or P 2 X 7 knockdown J774A.1 macrophages were stimulated with 1 μg/ml LPS for 6 h. The levels of NLRP3 and proIL-1β in the cell lysates were measured by Western blotting. The ELISA data are expressed as the mean ± SD of four separate experiments. The Western blotting results are representative of three different experiments. * and *** indicate significant differences at the levels of p < 0.05 and p < 0.001, respectively, compared to S. sonnei -infected cells (A–C) or untreated control cells (D) (one-way ANOVA with Dunnett's multiple comparisons test).

Article Snippet: Antibodies against IL-1β (AB-401-NA, polyclonal antibody) were purchased from R&D Systems (Minneapolis, MN).

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Western Blot, Knockdown, Flow Cytometry, Control

Journal: Frontiers in Immunology

Article Title: Critical Role for the NLRP3 Inflammasome in Mediating IL-1β Production in Shigella sonnei -Infected Macrophages

doi: 10.3389/fimmu.2020.01115

Figure Lengend Snippet: S. sonnei activates the NLRP3 inflammasome through H 2 O 2 production and lysosomal damage. (A) J774A.1 macrophages were primed with 1 μg/ml LPS for 4 h and then infected with 50 MOI S. sonnei for an additional 20 h. The levels of intracellular ROS were measured by CM-H 2 DCFDA staining, and the data were acquired by flow cytometry. (B) J774A.1 macrophages or BMDM were primed with 1 μg/ml LPS for 4 h and then treated with NAC for 0.5 h. The cells were then infected with 50 MOI S. sonnei for an additional 20 h. The levels of IL-1β and caspase-1 in the supernatants were measured by ELISA and Western blotting, respectively. (C) J774A.1 macrophages or BMDM were primed with 1 μg/ml LPS for 4 h and then treated with NDGA for 0.5 h. The cells were then infected with 50 MOI S. sonnei for an additional 20 h. The levels of IL-1β in the supernatants were measured by ELISA. (D,E) J774A.1 macrophages were primed with 1 μg/ml LPS for 4 h and then treated with NH 4 Cl and CQ (D) or CA-074-Me (E) for 0.5 h. The cells were then infected with 50 MOI S. sonnei for an additional 20 h. The levels of IL-1β in the supernatants were measured by ELISA. The ELISA data are expressed as the mean ± SD of four separate experiments. The flow cytometry and Western blotting results are representative of three different experiments. *, **, and *** indicate significant differences at the levels of p < 0.05, p < 0.01 and p < 0.001, respectively, compared to untreated control cells (A) or S. sonnei -infected cells (B–E) [two-tailed t -test in panel (A) ; one-way ANOVA with Dunnett's multiple comparisons test in panels (B–E) ].

Article Snippet: Antibodies against IL-1β (AB-401-NA, polyclonal antibody) were purchased from R&D Systems (Minneapolis, MN).

Techniques: Infection, Staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Western Blot, Control, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Critical Role for the NLRP3 Inflammasome in Mediating IL-1β Production in Shigella sonnei -Infected Macrophages

doi: 10.3389/fimmu.2020.01115

Figure Lengend Snippet: S. sonnei activates the NLRP3 inflammasome through mitochondrial damage. (A) J774A.1 macrophages were primed with 1 μg/ml LPS for 4 h and then treated with 20 μM MnTBAP for 0.5 h. The cells were then infected with 50 MOI S. sonnei for an additional 20 h. The levels of mitochondrial ROS were measured by MitoSOX staining and flow cytometry. (B) J774A.1 macrophages were primed with 1 μg/ml LPS for 4 h and then treated with MnTBAP for 0.5 h. The cells were then infected with 50 MOI S. sonnei for an additional 20 h. The levels of IL-1β in the supernatants were measured by ELISA. (C) J774A.1 macrophages were primed with 1 μg/ml LPS for 4 h and then infected with 50 MOI S. sonnei for an additional 20 h. The mitochondrial membrane potential was measured by DiOC 2 (3) staining and flow cytometry. (D) J774A.1 macrophages were primed with 1 μg/ml LPS for 4 h and then treated with cyclosporine A for 0.5 h. The cells were then infected with 50 MOI S. sonnei for an additional 20 h. The levels of IL-1β in the supernatants were measured by ELISA. The ELISA data are expressed as the mean ± SD of four separate experiments. The flow cytometry results are representative of three different experiments. ** and *** indicate significant differences at the levels of p < 0.01 and p < 0.001, respectively, compared to S. sonnei -infected cells (B,D) or as indicated (A,C) [two-tailed t -test in panels (A,C) ; one-way ANOVA with Dunnett's multiple comparisons test in panels (B,D) ].

Article Snippet: Antibodies against IL-1β (AB-401-NA, polyclonal antibody) were purchased from R&D Systems (Minneapolis, MN).

Techniques: Infection, Staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Membrane, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Critical Role for the NLRP3 Inflammasome in Mediating IL-1β Production in Shigella sonnei -Infected Macrophages

doi: 10.3389/fimmu.2020.01115

Figure Lengend Snippet: Phagocytosis of live S. sonnei is required for the full activation of the NLRP3 inflammasome. (A) J774A.1 macrophages were primed with 1 μg/ml LPS for 4 h and then treated with cytochalasin D for 0.5 h. The cells were then infected with 50 MOI S. sonnei for an additional 20 h. The levels of IL-1β and TNF-α in the supernatants were measured by ELISA. (B) The TLR2 mRNA expression of mock or TLR2 knockdown J774A.1 macrophages was measured by RT-qPCR. Mock or TLR2 knockdown J774A.1 macrophages were stimulated with 1 μg/ml Pam3CSK4 for 6 h. The levels of TNF-α in the supernatants were measured by ELISA. (C) Mock or TLR2 knockdown J774A.1 macrophages were primed with 1 μg/ml LPS for 4 h and then infected with 50 MOI live, heat-killed or freeze/thaw-killed S. sonnei for an additional 20 h. The levels of IL-1β and TNF-α in the supernatants were measured by ELISA. The ELISA data are expressed as the mean ± SD of four separate experiments. * and *** indicate significant differences at the levels of p < 0.05 and p < 0.001, respectively, compared to S. sonnei -infected cells (A) or live S. sonnei -infected cells (C) or as indicated (B) . ### indicates significant differences at the levels of p < 0.001 compared to sh-SC cells [one-way ANOVA with Dunnett's multiple comparisons test in panels (A,C) ; two-tailed t -test in panel (B) ].

Article Snippet: Antibodies against IL-1β (AB-401-NA, polyclonal antibody) were purchased from R&D Systems (Minneapolis, MN).

Techniques: Activation Assay, Infection, Enzyme-linked Immunosorbent Assay, Expressing, Knockdown, Quantitative RT-PCR, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Critical Role for the NLRP3 Inflammasome in Mediating IL-1β Production in Shigella sonnei -Infected Macrophages

doi: 10.3389/fimmu.2020.01115

Figure Lengend Snippet: NLRP3 knockout increases the bactericidal activity of macrophages against S. sonnei . (A,B) Wild-type or NLRP3-knockout J774A.1 macrophages were infected with 50 MOI S. sonnei for 15 min or 20 h. The cells were lysed, and the number of engulfed live S. sonnei cells was determined by the CFU assay and is indicated in the upper right corner (A) . The mean CFUs of cells infected with S. sonnei for 15 min or 20 h and the number of killed bacteria were calculated by subtracting the 20 h CFU assay result from the 15 min CFU assay result and are shown in panel (B) . The data are expressed as the mean ± SD of five separate experiments. ***indicates a significant difference at the level of p < 0.001 compared to wild-type cells (two-tailed t- test).

Article Snippet: Antibodies against IL-1β (AB-401-NA, polyclonal antibody) were purchased from R&D Systems (Minneapolis, MN).

Techniques: Knock-Out, Activity Assay, Infection, Colony-forming Unit Assay, Bacteria, Two Tailed Test

Journal: PLoS ONE

Article Title: Mesenchymal Transition and PDGFRA Amplification/Mutation Are Key Distinct Oncogenic Events in Pediatric Diffuse Intrinsic Pontine Gliomas

doi: 10.1371/journal.pone.0030313

Figure Lengend Snippet: Panel A: Radial plot showing the expression of oligodendroglial markers in the two groups of DIPG, in log2 ratios related each other. The red circle represent the median expression level of the whole population of DIPG. Group 1 expresses higher levels of oligodendroglial markers than group 2 DIPG. Panel B: Morphological oligodendroglial differenciation in group 1 tumors (HES staining, ×40). Panel C: Morphological astrocytic differenciation in group 2 tumors (HES staining ×40). Panel D: Olig2 immunohistochemistry in a group 1 DIPG showing that probably not all cells in the biopsy are tumoral (×40). Panel E: Dual immunohistochemistry for Olig2 and GFAP showing that tumor cells in mitosis are GFAP negative but Olig2 positive (×100). Panel F: Overall survival of 55 DIPG according to the presence (red) or absence (blue) of oligodendroglial differenciation. Median OS was shorter in patients with oligodendroglial type of tumors (7.73 vs 12.37, p = 0.045, log rank test).

Article Snippet: Sections were then incubated with various commercial monoclonal primary antibodies against Olig2 (AF 2418, 1/150, R/D system, CA, USA), P53 (DO-1, 1/1, Ventana) and MIB-1 (1/100; Dako, Glostrup, Denmark).

Techniques: Expressing, Staining, Immunohistochemistry

Journal: Journal of Inflammation (London, England)

Article Title: The PPAR-γ antagonist GW9662 elicits differentiation of M2c-like cells and upregulation of the MerTK/Gas6 axis: a key role for PPAR-γ in human macrophage polarization

doi: 10.1186/s12950-015-0081-4

Figure Lengend Snippet: GW9662 induces M2c-like cells that upregulate MerTK and its ligand Gas6. (A-C) Healthy monocytes were cultured in serum-free medium in the absence of cytokines or growth factors (M0 differentiation), with or without the PPAR-γ antagonist GW9662 (2.5-10 μM), for 4 days; when specified, the PPAR-γ agonist rosiglitazone (1 μM) was added. Expression of MerTK, CD163 and CD16 was measured by flow cytometry. (D-E) Gas6 production levels were quantified by ELISA in culture medium, upon incubation with or without GW9662 (2.5-10 μM) of otherwise untreated cells (M0 conditions), LPS (50 ng/ml; M1 conditions) or IL-4 (20 ng/ml; M2a conditions) exposed cells. (A-E) Pooled data are represented as mean values ± SEM. Analysis was performed using one-way repeated measures ANOVA with Newman-Keuls multiple comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. When not specified by additional graphic signs, statistical annotations (asterisks) refer to comparisons with respect to the relative GW9662 untreated control group. Each set of data is representative of three independent experiments.

Article Snippet: Biotinylated goat polyclonal anti-human Gas6 antibody (R&D Systems), followed by HRP-conjugated streptavidin (Biolegend), was used for detection.

Techniques: Cell Culture, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Incubation, Control