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MedChemExpress purchase anti human neutralizing il 6 antibody
Purchase Anti Human Neutralizing Il 6 Antibody, supplied by MedChemExpress, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Differential Effects of vIL6 and hIL6 on the frequency and distribution of KSHV infection in B cells. Naïve B cells were isolated from 15 unique tonsils and Mock infected or infected with BAC16‐derived KSHV‐WT or KSHV‐ΔK2. Cultures were treated with IL6 neutralizing antibodies or left untreated and analyzed at 3 dpi for the distribution of KSHV infection within B cell lineages (Table ) using the GFP reporter by flow cytometry. (A) percent of viable CD19 + B lymphocytes that were GFP+ in each condition. Two‐way repeated measures ANOVA p = 0.006, F = 10.6 for Nab treatment, post hoc paired T ‐test p = 0.04 comparing no treatment to IL6 neutralization in KSHV‐WT infection. (B) Percent of GFP+ plasmablasts; paired T ‐test p = 0.05 comparing WT to ∆K2 in untreated cultures (C) percent of GFP+ classical memory cells; Two‐way repeated measures ANOVA p = 0.03, F = 5.7 for Nab treatment, p = 0.03, F = 5.9 for virus strain; paired T ‐test p = 0.04 comparing WT to ∆K2 in untreated cultures. (D) Percent of GFP + IL6 + CD19+ cells; paired T ‐test p = 0.03 comparing WT and ∆K2 infection in the untreated cultures. (E) Correlation between total infection (GFP + CD19 + ) and GFP within plasmablast using pearson's method. (F) Correlation between total infection (GFP + CD19 + ) and GFP within classical memory using pearson's method. (G) lack of correlation between total infection (GFP + CD19 + ) and GFP within IL6 + B cells. (H) Percent of GFP + MZ‐like cells; Two‐way repeated measures ANOVA p = 0.006, F = 10.6 for Nab treatment, post hoc paired T ‐test p = 0.03 comparing no treatment to IL6 neutralization in KSHV‐∆K2 infection. (I) Correlation between total infection (GFP + CD19 + ) and GFP within MZ‐like using pearson's method.

Journal: Journal of Medical Virology

Article Title: KSHV vIL6 Inhibits Functional B Cell Maturation During De Novo Infection

doi: 10.1002/jmv.70479

Figure Lengend Snippet: Differential Effects of vIL6 and hIL6 on the frequency and distribution of KSHV infection in B cells. Naïve B cells were isolated from 15 unique tonsils and Mock infected or infected with BAC16‐derived KSHV‐WT or KSHV‐ΔK2. Cultures were treated with IL6 neutralizing antibodies or left untreated and analyzed at 3 dpi for the distribution of KSHV infection within B cell lineages (Table ) using the GFP reporter by flow cytometry. (A) percent of viable CD19 + B lymphocytes that were GFP+ in each condition. Two‐way repeated measures ANOVA p = 0.006, F = 10.6 for Nab treatment, post hoc paired T ‐test p = 0.04 comparing no treatment to IL6 neutralization in KSHV‐WT infection. (B) Percent of GFP+ plasmablasts; paired T ‐test p = 0.05 comparing WT to ∆K2 in untreated cultures (C) percent of GFP+ classical memory cells; Two‐way repeated measures ANOVA p = 0.03, F = 5.7 for Nab treatment, p = 0.03, F = 5.9 for virus strain; paired T ‐test p = 0.04 comparing WT to ∆K2 in untreated cultures. (D) Percent of GFP + IL6 + CD19+ cells; paired T ‐test p = 0.03 comparing WT and ∆K2 infection in the untreated cultures. (E) Correlation between total infection (GFP + CD19 + ) and GFP within plasmablast using pearson's method. (F) Correlation between total infection (GFP + CD19 + ) and GFP within classical memory using pearson's method. (G) lack of correlation between total infection (GFP + CD19 + ) and GFP within IL6 + B cells. (H) Percent of GFP + MZ‐like cells; Two‐way repeated measures ANOVA p = 0.006, F = 10.6 for Nab treatment, post hoc paired T ‐test p = 0.03 comparing no treatment to IL6 neutralization in KSHV‐∆K2 infection. (I) Correlation between total infection (GFP + CD19 + ) and GFP within MZ‐like using pearson's method.

Article Snippet: IL6 neutralizing antibodies were obtained from R&D Systems (7270‐IL‐010/CF).

Techniques: Infection, Isolation, Derivative Assay, Flow Cytometry, Neutralization, Virus

vIL6 impacts hIL6 expression in B cell subsets. Naïve B cells were isolated from 15 unique tonsils and Mock infected or infected with BAC16‐derived KSHV‐WT or KSHV‐ΔK2. Cultures were treated with IL6 neutralizing antibodies or left untreated. At 3 dpi supernatants were analyzed for secreted hIL6 and cells were analyzed for hIL6 expression within B cell subsets by ICCS. (A) Frequency of IL6 + B cells. Two‐way repeated measures ANOVA p = 0.002, F = 7.6 for virus strain, post hoc paired T ‐test p = 0.006 comparing KSHV‐WT to ∆K2 in the no treatment condition. (B) Secreted hIL6 showed the same statistical effect as IL6+ within B cells ( p = 0.01). (C) Frequency of IL6+ within B cell subsets with the following significant effects. Plasma cell: Two‐way repeated measures ANOVA p = 0.02, F = 4.4 for virus strain, post hoc paired T ‐test p = 0.05 comparing KSHV‐WT to ∆K2 in the hIL6 neutralized condition. Double negative memory: paired T ‐test p = 0.0 comparing KSHV‐WT to ∆K2 in the no treatment condition. Naive: Two‐way repeated measures ANOVA p = 0.03, F = 3.9 for virus strain, post hoc paired T ‐test p = 0.02 comparing KSHV‐WT to ∆K2 in the no treatment condition. Transitional: Two‐way repeated measures ANOVA p = 0.003, F = 7.3 for virus strain, post hoc paired T ‐test p = 0.04 comparing KSHV‐WT to ∆K2 in the no treatment condition. Germinal Center: Two‐way repeated measures ANOVA p = 0.002, F = 7.7 for virus strain, post hoc paired T ‐tests p = 0.02 comparing KSHV‐WT to ∆K2 in the hIL6 neutralized condition, p = 0.01 comparing KSHV‐WT to ∆K2 in the no treatment condition.

Journal: Journal of Medical Virology

Article Title: KSHV vIL6 Inhibits Functional B Cell Maturation During De Novo Infection

doi: 10.1002/jmv.70479

Figure Lengend Snippet: vIL6 impacts hIL6 expression in B cell subsets. Naïve B cells were isolated from 15 unique tonsils and Mock infected or infected with BAC16‐derived KSHV‐WT or KSHV‐ΔK2. Cultures were treated with IL6 neutralizing antibodies or left untreated. At 3 dpi supernatants were analyzed for secreted hIL6 and cells were analyzed for hIL6 expression within B cell subsets by ICCS. (A) Frequency of IL6 + B cells. Two‐way repeated measures ANOVA p = 0.002, F = 7.6 for virus strain, post hoc paired T ‐test p = 0.006 comparing KSHV‐WT to ∆K2 in the no treatment condition. (B) Secreted hIL6 showed the same statistical effect as IL6+ within B cells ( p = 0.01). (C) Frequency of IL6+ within B cell subsets with the following significant effects. Plasma cell: Two‐way repeated measures ANOVA p = 0.02, F = 4.4 for virus strain, post hoc paired T ‐test p = 0.05 comparing KSHV‐WT to ∆K2 in the hIL6 neutralized condition. Double negative memory: paired T ‐test p = 0.0 comparing KSHV‐WT to ∆K2 in the no treatment condition. Naive: Two‐way repeated measures ANOVA p = 0.03, F = 3.9 for virus strain, post hoc paired T ‐test p = 0.02 comparing KSHV‐WT to ∆K2 in the no treatment condition. Transitional: Two‐way repeated measures ANOVA p = 0.003, F = 7.3 for virus strain, post hoc paired T ‐test p = 0.04 comparing KSHV‐WT to ∆K2 in the no treatment condition. Germinal Center: Two‐way repeated measures ANOVA p = 0.002, F = 7.7 for virus strain, post hoc paired T ‐tests p = 0.02 comparing KSHV‐WT to ∆K2 in the hIL6 neutralized condition, p = 0.01 comparing KSHV‐WT to ∆K2 in the no treatment condition.

Article Snippet: IL6 neutralizing antibodies were obtained from R&D Systems (7270‐IL‐010/CF).

Techniques: Expressing, Isolation, Infection, Derivative Assay, Virus, Clinical Proteomics

vIL6 and hIL6 differentially alter secretion of TNF‐α, BAFF and IL‐10 during KSHV infection. Supernatants from 11 unique tonsils infected and treated as in experiments described in Figure were harvested at 3 dpi and clarified of cellular debris by centrifugation. Concentrations of 13 cytokines related to B cell activation and differentiation were determined using the Biolegend Legendplex Human B cell panel for each supernatant. Cytokines significantly altered by experimental parameters were (A) BAFF with 2‐way repeated measures ANOVA showing significant interaction of virus infection and hIL‐6 neutralization ( p = 0.03, F = 4.4) and significantly different pairwise comparison via T‐test comparing Mock+IL6 NAb and KSHV‐∆K2 + IL6 Nab ( p = 0.039). (B) TNF‐α with 2‐way repeated measures ANOVA showing significant effect of virus condition ( p = 0.05, F = 3.6) and significant pairwise comparisons via T ‐test comparing Mock and KSHV‐WT ( p = 0.008) and KSHV‐WT with KSHV‐∆K2 ( p = 0.01) with no hIL6 neutralization; (C) IL‐10 with significant pairwise comparisons via T‐test comparing Mock and KSHV‐WT ( p = 0.02) with no hIL6 neutralization.

Journal: Journal of Medical Virology

Article Title: KSHV vIL6 Inhibits Functional B Cell Maturation During De Novo Infection

doi: 10.1002/jmv.70479

Figure Lengend Snippet: vIL6 and hIL6 differentially alter secretion of TNF‐α, BAFF and IL‐10 during KSHV infection. Supernatants from 11 unique tonsils infected and treated as in experiments described in Figure were harvested at 3 dpi and clarified of cellular debris by centrifugation. Concentrations of 13 cytokines related to B cell activation and differentiation were determined using the Biolegend Legendplex Human B cell panel for each supernatant. Cytokines significantly altered by experimental parameters were (A) BAFF with 2‐way repeated measures ANOVA showing significant interaction of virus infection and hIL‐6 neutralization ( p = 0.03, F = 4.4) and significantly different pairwise comparison via T‐test comparing Mock+IL6 NAb and KSHV‐∆K2 + IL6 Nab ( p = 0.039). (B) TNF‐α with 2‐way repeated measures ANOVA showing significant effect of virus condition ( p = 0.05, F = 3.6) and significant pairwise comparisons via T ‐test comparing Mock and KSHV‐WT ( p = 0.008) and KSHV‐WT with KSHV‐∆K2 ( p = 0.01) with no hIL6 neutralization; (C) IL‐10 with significant pairwise comparisons via T‐test comparing Mock and KSHV‐WT ( p = 0.02) with no hIL6 neutralization.

Article Snippet: IL6 neutralizing antibodies were obtained from R&D Systems (7270‐IL‐010/CF).

Techniques: Infection, Centrifugation, Activation Assay, Virus, Neutralization, Comparison

vIL6 and hIL6 cooperatively support B cell viability during KSHV infection but have differential effects on subset frequencies. Naïve B cells were extracted from a distinct set of 15 tonsils and subjected to infection with either Mock, KSHV‐WT, or KSHV‐ΔK2. The cultures were either supplemented with IL6‐neutralizing antibodies or remained untreated. At 3dpi, the cultures were examined by flow cytometry to assess B cell viabilities and the frequencies of B cell subsets. (A) Percent of viable CD19 + . Two‐way repeated measures ANOVA p = 0.01, F = 5.3 for virus strain, p = 0.04, F = 5.5 for hIL6 neutralization and p = 0.03, F = 3.9 for the interaction of the two variables. Post hoc paired T ‐tests with Holm correction for multiple comparisons showed p = 0.01 comparing no treatment to hIL6 neutralization in Mock cultures, p = 0.01 comparing Mock with KSHV‐WT in untreated cultures and p = 0.004 comparin Mock with ∆K2 in untreated cultures. (B) Frequency of naïve B cells Two‐way repeated measures ANOVA p = 0.05, F = 4.7 for hIL6 neutralization and p = 0.04, F = 3.6 for the interaction of virus and hIL6 neutralization. Post hoc paired T ‐tests with Holm correction for multiple comparisons showed p = 0.018 comparing Mock to ∆K2 in the no treatment condition and p = 0.01 comparing untreated to hIL6 neutralization in Mock infected cultures. (C) Frequency of germinal center B cells. Two‐way repeated measures ANOVA p = 0.03, F = 4.1 for virus strain. Post hoc paired T ‐tests with Holm correction for multiple comparisons showed p = 0.019 comparing Mock to ∆K2 in the no treatment condition. (D) Frequency of plasmablasts. Two‐way repeated measures ANOVA p = 0.00006, F = 14.1 for virus strain and p = 0.001, F = 9.0 for the interaction of virus and hIL6 neutralization. Post hoc paired T ‐tests with Holm correction for multiple comparisons showed p = 0.03 comparing Mock and WT, p = 0.03 comparing Mock and ∆K2 in the no treatment conditions and p = 0.002 comparing Mock and WT, p = 0.04 comparing Mock and ∆K2 and p = 0.004 comparing WT and ∆K2 in the hIL6 neutralized conditions.

Journal: Journal of Medical Virology

Article Title: KSHV vIL6 Inhibits Functional B Cell Maturation During De Novo Infection

doi: 10.1002/jmv.70479

Figure Lengend Snippet: vIL6 and hIL6 cooperatively support B cell viability during KSHV infection but have differential effects on subset frequencies. Naïve B cells were extracted from a distinct set of 15 tonsils and subjected to infection with either Mock, KSHV‐WT, or KSHV‐ΔK2. The cultures were either supplemented with IL6‐neutralizing antibodies or remained untreated. At 3dpi, the cultures were examined by flow cytometry to assess B cell viabilities and the frequencies of B cell subsets. (A) Percent of viable CD19 + . Two‐way repeated measures ANOVA p = 0.01, F = 5.3 for virus strain, p = 0.04, F = 5.5 for hIL6 neutralization and p = 0.03, F = 3.9 for the interaction of the two variables. Post hoc paired T ‐tests with Holm correction for multiple comparisons showed p = 0.01 comparing no treatment to hIL6 neutralization in Mock cultures, p = 0.01 comparing Mock with KSHV‐WT in untreated cultures and p = 0.004 comparin Mock with ∆K2 in untreated cultures. (B) Frequency of naïve B cells Two‐way repeated measures ANOVA p = 0.05, F = 4.7 for hIL6 neutralization and p = 0.04, F = 3.6 for the interaction of virus and hIL6 neutralization. Post hoc paired T ‐tests with Holm correction for multiple comparisons showed p = 0.018 comparing Mock to ∆K2 in the no treatment condition and p = 0.01 comparing untreated to hIL6 neutralization in Mock infected cultures. (C) Frequency of germinal center B cells. Two‐way repeated measures ANOVA p = 0.03, F = 4.1 for virus strain. Post hoc paired T ‐tests with Holm correction for multiple comparisons showed p = 0.019 comparing Mock to ∆K2 in the no treatment condition. (D) Frequency of plasmablasts. Two‐way repeated measures ANOVA p = 0.00006, F = 14.1 for virus strain and p = 0.001, F = 9.0 for the interaction of virus and hIL6 neutralization. Post hoc paired T ‐tests with Holm correction for multiple comparisons showed p = 0.03 comparing Mock and WT, p = 0.03 comparing Mock and ∆K2 in the no treatment conditions and p = 0.002 comparing Mock and WT, p = 0.04 comparing Mock and ∆K2 and p = 0.004 comparing WT and ∆K2 in the hIL6 neutralized conditions.

Article Snippet: IL6 neutralizing antibodies were obtained from R&D Systems (7270‐IL‐010/CF).

Techniques: Infection, Flow Cytometry, Virus, Neutralization

$hIL6 and vIL6 have separable effects on the differentiation of plasmablast and germinal center B cells from naïve B cells during infection. I B cells loaded with tracking dye before infection and coculture with unlabeled lymphocyte fractions to track differentiation of naïve B cells over time during Mock, KSHV‐WT or KSHV‐∆K2 infection with or without neutralization of hIL6. At 3 dpi cells were harvested and analyzed for B cell subset markers, KSHV infection (GFP+ populations) and differentiation (dye+ populations). (A) proportional distribution of dye+ B cells amongst B cell subsets (B) frequency of undifferentiated naïve B cells. Two‐way repeated measures ANOVA p = 0.02, F = 3.6 for interaction of virus strain and hIL6 neutralization. Post‐hoc paired T ‐test p = 0.031 comparing Mock to ∆K2 in the untreated condition. (C) Frequency of GC B cells differentiated from naïve. Two‐way repeated measures ANOVA p = 0.02, F = 3.6 for virus strain, p = 0.03, F = 3.8 for interaction of virus strain and hIL6 neutralization. Post‐hoc paired T ‐test p = 0.039 comparing Mock to ∆K2 and p = 0.035 comparing WT to ∆K2 in the untreated conditions. (D) Frequency of plasmablast differentiated from naïve. Two‐way repeated measures ANOVA p = 0.00004, F = 10.2 for virus strain, p = 0.05, F = 3.9 for interaction of virus strain and hIL6 neutralization. Post‐hoc paired T ‐test p = 0.012 comparing Mock to ∆K2 and p = 0.014 comparing Mock to WT in the untreated conditions, p = 0.007 comparing Mock to WT and p = 0.011 comparing WT to ∆K2 in the hIL6 neutralized conditions. (E) Correlation between differentiation of GCB and total frequency of GCB using pearson's method. (F) Correlation between differentiation of plasmablast and total frequency of plasmablast via pearson's method. (G) Correlation between differentiation of GCB and IL6 + GCB within each virus condition using pearson's method. (H) Correlation between GCB differentiation and total KSHV infection using pearson's method. (I) Correlation between differentiation of GCB and KSHV infection of plasmablast using pearson's method. (J) Correlation between differentiation of GCB and KSHV infection of classical memory using pearson's method.$

Journal: Journal of Medical Virology

Article Title: KSHV vIL6 Inhibits Functional B Cell Maturation During De Novo Infection

doi: 10.1002/jmv.70479

Figure Lengend Snippet: hIL6 and vIL6 have separable effects on the differentiation of plasmablast and germinal center B cells from naïve B cells during infection. I B cells loaded with tracking dye before infection and coculture with unlabeled lymphocyte fractions to track differentiation of naïve B cells over time during Mock, KSHV‐WT or KSHV‐∆K2 infection with or without neutralization of hIL6. At 3 dpi cells were harvested and analyzed for B cell subset markers, KSHV infection (GFP+ populations) and differentiation (dye+ populations). (A) proportional distribution of dye+ B cells amongst B cell subsets (B) frequency of undifferentiated naïve B cells. Two‐way repeated measures ANOVA p = 0.02, F = 3.6 for interaction of virus strain and hIL6 neutralization. Post‐hoc paired T ‐test p = 0.031 comparing Mock to ∆K2 in the untreated condition. (C) Frequency of GC B cells differentiated from naïve. Two‐way repeated measures ANOVA p = 0.02, F = 3.6 for virus strain, p = 0.03, F = 3.8 for interaction of virus strain and hIL6 neutralization. Post‐hoc paired T ‐test p = 0.039 comparing Mock to ∆K2 and p = 0.035 comparing WT to ∆K2 in the untreated conditions. (D) Frequency of plasmablast differentiated from naïve. Two‐way repeated measures ANOVA p = 0.00004, F = 10.2 for virus strain, p = 0.05, F = 3.9 for interaction of virus strain and hIL6 neutralization. Post‐hoc paired T ‐test p = 0.012 comparing Mock to ∆K2 and p = 0.014 comparing Mock to WT in the untreated conditions, p = 0.007 comparing Mock to WT and p = 0.011 comparing WT to ∆K2 in the hIL6 neutralized conditions. (E) Correlation between differentiation of GCB and total frequency of GCB using pearson's method. (F) Correlation between differentiation of plasmablast and total frequency of plasmablast via pearson's method. (G) Correlation between differentiation of GCB and IL6 + GCB within each virus condition using pearson's method. (H) Correlation between GCB differentiation and total KSHV infection using pearson's method. (I) Correlation between differentiation of GCB and KSHV infection of plasmablast using pearson's method. (J) Correlation between differentiation of GCB and KSHV infection of classical memory using pearson's method.

Article Snippet: IL6 neutralizing antibodies were obtained from R&D Systems (7270‐IL‐010/CF).

Techniques: Infection, Neutralization, Virus

$KSHV infection drives functional maturation of B cells during ex vivo infection, but vIL6 suppresses differentiation of IgG+ plasma cells during KSHV infection. Naive B cells loaded with tracking dye before infection and coculture with unlabeled lymphocyte fractions to track differentiation of naïve B cells over time during Mock, KSHV‐WT or KSHV‐∆K2 infection with or without neutralization of hIL6. At 3 dpi cells were harvested and analyzed for B cell subset markers including Ig isotype, KSHV infection (GFP+ populations) and differentiation (dye+ populations). (A) Frequency of differentiated IgG+ B cells normalized to each sample's mock value. Two‐way repeated measures ANOVA conducted before data normalization: p = 0.00008, F = 15.4 for virus strain, p = 0.04, F = 5.1 for hIL6 neutralization and p = 0.002, F = 7.4 for interaction of virus strain and hIL6 neutralization. (B) frequency of differentiated IgG+ plasma cells. Paired T ‐test p = 0.03 comparing WT and ∆K2 in the untreated condition. (C) Concentrations of IgG isotypes at 3 dpi were determined in clarified supernatants from eight samples via Legendplex immunoassay. Two‐way repeated measures ANOVA showed significant effect of virus strain on supernatant concentrations of IgG1 ( p = 0.036, F = 6.4) and significant effect of virus strain ( p = 0.04, F = 5.99) and significant interaction of virus strain and IL6 neutralization ( p = 0.04, F = 6) on supernatant concentrations of IgG3.$

Journal: Journal of Medical Virology

Article Title: KSHV vIL6 Inhibits Functional B Cell Maturation During De Novo Infection

doi: 10.1002/jmv.70479

Figure Lengend Snippet: KSHV infection drives functional maturation of B cells during ex vivo infection, but vIL6 suppresses differentiation of IgG+ plasma cells during KSHV infection. Naive B cells loaded with tracking dye before infection and coculture with unlabeled lymphocyte fractions to track differentiation of naïve B cells over time during Mock, KSHV‐WT or KSHV‐∆K2 infection with or without neutralization of hIL6. At 3 dpi cells were harvested and analyzed for B cell subset markers including Ig isotype, KSHV infection (GFP+ populations) and differentiation (dye+ populations). (A) Frequency of differentiated IgG+ B cells normalized to each sample's mock value. Two‐way repeated measures ANOVA conducted before data normalization: p = 0.00008, F = 15.4 for virus strain, p = 0.04, F = 5.1 for hIL6 neutralization and p = 0.002, F = 7.4 for interaction of virus strain and hIL6 neutralization. (B) frequency of differentiated IgG+ plasma cells. Paired T ‐test p = 0.03 comparing WT and ∆K2 in the untreated condition. (C) Concentrations of IgG isotypes at 3 dpi were determined in clarified supernatants from eight samples via Legendplex immunoassay. Two‐way repeated measures ANOVA showed significant effect of virus strain on supernatant concentrations of IgG1 ( p = 0.036, F = 6.4) and significant effect of virus strain ( p = 0.04, F = 5.99) and significant interaction of virus strain and IL6 neutralization ( p = 0.04, F = 6) on supernatant concentrations of IgG3.

Article Snippet: IL6 neutralizing antibodies were obtained from R&D Systems (7270‐IL‐010/CF).

Techniques: Infection, Functional Assay, Ex Vivo, Clinical Proteomics, Neutralization, Virus

circCUL2 regulates the phenotypic plasticity of CAFs and promote PDAC progression by IL6. A Gene Set Enrichment Analysis (GSEA) of affected signatures in fibroblasts (circCUL2 vs. control). B GSEA plots for inflammatory CAF (iCAF) signatures in circCUL2 overexpression NFs from control. C - D qRT–PCR analysis of iCAF markers (IL6, TNF-α and IL1α) and myCAF marker (Acta2 and Axin2) expression in circCUL2 overexpression NFs. E Flow cytometric analysis of PDGFRα and α-SMA expression in circCUL2 overexpression NFs. F - H . Representative cytokine arrays for circCUL2-overexpression NFs and control (n = 3). arrows indicate the cytokines with significant changes, which were further confirmed by qRT–PCR and ELISA. I - L EdU assay ( I ), colony formation ( J ), Scratch wound healing assays ( K ) and transwell assays ( L ) of PANC-1 cells treated with conditioned medium circCUL2-overexpression NFs or anti-IL6. Scale bar, 100 μm. M western blot analysis of STAT3 and p-STAT3 in PANC-1 cells. Data are expressed as the mean ± SD of three independent experiments. ** p < 0.01 and *** p < 0.001 (two-tailed Student t-tests)

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: circCUL2 induces an inflammatory CAF phenotype in pancreatic ductal adenocarcinoma via the activation of the MyD88-dependent NF-κB signaling pathway

doi: 10.1186/s13046-021-02237-6

Figure Lengend Snippet: circCUL2 regulates the phenotypic plasticity of CAFs and promote PDAC progression by IL6. A Gene Set Enrichment Analysis (GSEA) of affected signatures in fibroblasts (circCUL2 vs. control). B GSEA plots for inflammatory CAF (iCAF) signatures in circCUL2 overexpression NFs from control. C - D qRT–PCR analysis of iCAF markers (IL6, TNF-α and IL1α) and myCAF marker (Acta2 and Axin2) expression in circCUL2 overexpression NFs. E Flow cytometric analysis of PDGFRα and α-SMA expression in circCUL2 overexpression NFs. F - H . Representative cytokine arrays for circCUL2-overexpression NFs and control (n = 3). arrows indicate the cytokines with significant changes, which were further confirmed by qRT–PCR and ELISA. I - L EdU assay ( I ), colony formation ( J ), Scratch wound healing assays ( K ) and transwell assays ( L ) of PANC-1 cells treated with conditioned medium circCUL2-overexpression NFs or anti-IL6. Scale bar, 100 μm. M western blot analysis of STAT3 and p-STAT3 in PANC-1 cells. Data are expressed as the mean ± SD of three independent experiments. ** p < 0.01 and *** p < 0.001 (two-tailed Student t-tests)

Article Snippet: The mice were randomly divided into three groups, which were injected with luc-PANC-1 or luc-MiaPaCa-2 incubated with conditioned medium from (1) NFs transduced with empty vector for 48 h, (2) NFs transduced with circCUL2 vector for 48 h, (3) NFs transduced with circCUL2 vector in the presence of IL6 neutralizing antibodies (50 ng/mL, MAB206, R&D) for 48 h. The mice were imaged with an In Vivo Imaging System (IVIS Lumina XR Series III) 30 days later.

Techniques: Control, Over Expression, Quantitative RT-PCR, Marker, Expressing, Enzyme-linked Immunosorbent Assay, EdU Assay, Western Blot, Two Tailed Test

circCUL2-overexpression NFs promote PDAC progression in vivo. A Representative Bioluminescence images, lung and HE staining of lung tissue of mice 4 weeks after tail vein injection of luc-PANC-1 cell treated with conditioned medium as indicated (n = 8 per group). Scale bar, 100 μm. B Relative luminescence intensity in each group. C Histogram analysis of the metastatic nodules number in per lung. D lung metastasis rate of each group (Chi-square test). E - F Representative bioluminescence images and histogram analysis of luminescence intensity in each at day 30 are shown (n = 6). G Abdominal metastasis rate was calculated for indicated group (Chi-square test). H Representative images of orthotopic model in each group on which autopsy was performed. Red arrow indicated primary tumor; S, spleen; T, primary tumor; M, metastasis. I Images of PDX from 2 patients in 5 mice. ( J ) Tumor growth curves of indicated group (n = 5). K qRT–PCR analysis of circCUL2 levels in PDX of mice before and after treatment. L Representative images of IHC for PDGFRα and IL6. Scale bar, 100 μm. Data are expressed as the mean ± SD. ** p < 0.01 and *** p < 0.001 (two-tailed Student t-tests)

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: circCUL2 induces an inflammatory CAF phenotype in pancreatic ductal adenocarcinoma via the activation of the MyD88-dependent NF-κB signaling pathway

doi: 10.1186/s13046-021-02237-6

Figure Lengend Snippet: circCUL2-overexpression NFs promote PDAC progression in vivo. A Representative Bioluminescence images, lung and HE staining of lung tissue of mice 4 weeks after tail vein injection of luc-PANC-1 cell treated with conditioned medium as indicated (n = 8 per group). Scale bar, 100 μm. B Relative luminescence intensity in each group. C Histogram analysis of the metastatic nodules number in per lung. D lung metastasis rate of each group (Chi-square test). E - F Representative bioluminescence images and histogram analysis of luminescence intensity in each at day 30 are shown (n = 6). G Abdominal metastasis rate was calculated for indicated group (Chi-square test). H Representative images of orthotopic model in each group on which autopsy was performed. Red arrow indicated primary tumor; S, spleen; T, primary tumor; M, metastasis. I Images of PDX from 2 patients in 5 mice. ( J ) Tumor growth curves of indicated group (n = 5). K qRT–PCR analysis of circCUL2 levels in PDX of mice before and after treatment. L Representative images of IHC for PDGFRα and IL6. Scale bar, 100 μm. Data are expressed as the mean ± SD. ** p < 0.01 and *** p < 0.001 (two-tailed Student t-tests)

Techniques: Over Expression, In Vivo, Staining, Injection, Quantitative RT-PCR, Two Tailed Test

MyD88 is a direct downstream target gene of miR-203a-3p. A - B Colony formation and transwell assays of PANC-1 cells treated with conditioned medium from miR-203a-3p-silencing NFs or miR-203a-3p-overexpression CAFs. Scale bar: 100 μm. C - D ELISA assays detected IL6 level of conditioned medium from miR-203a-3p-silencing NFs or miR-203a-3p-overexpression CAFs. E Venn analysis of the potential downstream target genes of miR-203a-3p, predicted by miRTarbase, miRWalk and Tarbase. F - G qRT–PCR analysis of screened downstream target genes of miR-203a-3p in indicated NFs and CAFs. H Luciferase reporter assay was used to detect the luciferase activity of MyD88-Wild Type (MyD88-wt) and miR-203a-3p binding site mutated MyD88 (mYD88-mut) luciferase reporter cotransfected with miR-203a-3p mimic. N.S., no significant. I - J Western blotting analysis of MyD88, p65, pp65, IKBα and p-IKBα expression after overexpression of circCUL2 or silence of miR-203a-3p in NFs, or silence of circCUL2 or overexpression of miR-203a-3p in CAFs. GAPDH as a loading control. Data are expressed as the mean ± SD. ** p < 0.01 and *** p < 0.001

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: circCUL2 induces an inflammatory CAF phenotype in pancreatic ductal adenocarcinoma via the activation of the MyD88-dependent NF-κB signaling pathway

doi: 10.1186/s13046-021-02237-6

Figure Lengend Snippet: MyD88 is a direct downstream target gene of miR-203a-3p. A - B Colony formation and transwell assays of PANC-1 cells treated with conditioned medium from miR-203a-3p-silencing NFs or miR-203a-3p-overexpression CAFs. Scale bar: 100 μm. C - D ELISA assays detected IL6 level of conditioned medium from miR-203a-3p-silencing NFs or miR-203a-3p-overexpression CAFs. E Venn analysis of the potential downstream target genes of miR-203a-3p, predicted by miRTarbase, miRWalk and Tarbase. F - G qRT–PCR analysis of screened downstream target genes of miR-203a-3p in indicated NFs and CAFs. H Luciferase reporter assay was used to detect the luciferase activity of MyD88-Wild Type (MyD88-wt) and miR-203a-3p binding site mutated MyD88 (mYD88-mut) luciferase reporter cotransfected with miR-203a-3p mimic. N.S., no significant. I - J Western blotting analysis of MyD88, p65, pp65, IKBα and p-IKBα expression after overexpression of circCUL2 or silence of miR-203a-3p in NFs, or silence of circCUL2 or overexpression of miR-203a-3p in CAFs. GAPDH as a loading control. Data are expressed as the mean ± SD. ** p < 0.01 and *** p < 0.001

Techniques: Over Expression, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Luciferase, Reporter Assay, Activity Assay, Binding Assay, Western Blot, Expressing, Control

circCUL2 promotes proliferation and migration via MyD88/NF-κB/IL6 axis. A - D Cotransfection of circCUL2 overexpression plasmid and miR-203a-3p mimic in NFs or circCUL2 siRNA and miR-203-3p inhibitor in CAFs to detect the protein level of MyD88, p65, pp65, IKBα and p-IKBα, and secretion level of IL6. E - F PANC-1 cells were treated with conditioned medium from NFs cotransfected circCUL2 overexpression plasmid with miR-203a-3p mimic, or CAFs cotransfected circCUL2 siRNA and miR-203-3p inhibitor for 48 h. The proliferation and migration ability of PANC-1 were detected by colony formation and transwell assays. G - J Cotransfection of circCUL2 overexpression plasmid and MyD88 siRNA in NFs or circCUL2 siRNA and MyD88 overexpression plasmid in CAFs to detect the protein level of MyD88, p65, pp65, IKBα and p-IKBα and secretion level of IL6. K - L PANC-1 cells were treated with conditioned medium from NFs cotransfected circCUL2 overexpression plasmid with MyD88 siRNA, or CAFs cotransfected circCUL2 siRNA and MyD88 overexpression plasmid for 48 h. The proliferation and migration ability of PANC-1 were detected by colony formation and transwell assays. Data are expressed as the mean ± SD. ** p < 0.01 and *** p < 0.001

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: circCUL2 induces an inflammatory CAF phenotype in pancreatic ductal adenocarcinoma via the activation of the MyD88-dependent NF-κB signaling pathway

doi: 10.1186/s13046-021-02237-6

Figure Lengend Snippet: circCUL2 promotes proliferation and migration via MyD88/NF-κB/IL6 axis. A - D Cotransfection of circCUL2 overexpression plasmid and miR-203a-3p mimic in NFs or circCUL2 siRNA and miR-203-3p inhibitor in CAFs to detect the protein level of MyD88, p65, pp65, IKBα and p-IKBα, and secretion level of IL6. E - F PANC-1 cells were treated with conditioned medium from NFs cotransfected circCUL2 overexpression plasmid with miR-203a-3p mimic, or CAFs cotransfected circCUL2 siRNA and miR-203-3p inhibitor for 48 h. The proliferation and migration ability of PANC-1 were detected by colony formation and transwell assays. G - J Cotransfection of circCUL2 overexpression plasmid and MyD88 siRNA in NFs or circCUL2 siRNA and MyD88 overexpression plasmid in CAFs to detect the protein level of MyD88, p65, pp65, IKBα and p-IKBα and secretion level of IL6. K - L PANC-1 cells were treated with conditioned medium from NFs cotransfected circCUL2 overexpression plasmid with MyD88 siRNA, or CAFs cotransfected circCUL2 siRNA and MyD88 overexpression plasmid for 48 h. The proliferation and migration ability of PANC-1 were detected by colony formation and transwell assays. Data are expressed as the mean ± SD. ** p < 0.01 and *** p < 0.001

Techniques: Migration, Cotransfection, Over Expression, Plasmid Preparation

Clinical implication of circCUL2/miR-203a-3p/IL6 axis in PDAC. A , F , K The relative expression of miR-203a-3p ( A ), MyD88 ( F ) and IL6 ( G ) in 161 PDAC tissues compared to paired NAT. B - C , G - H , L - M Association analysis between miR-203a-3p ( B - C ), MyD88 ( G - H ) and IL6 ( L - M ) expression levels and LN status and tumor stages in 161 PDAC tissues. ( D - E , I - J , N - O ) Kaplan-Meier analysis of the correlation between miR-203a-3p ( D - E ), MyD88 ( I - J ) and IL6 ( N - O ) expression levels and OS or DFS of 161 PDAC patients. The median miR-203a-3p, MyD88 and IL6 expression was used as the cutoff value. P Proposed model indicates the mechanism by which circCUL2 activated iCAF phenotype and production of IL6 to promote malignant progression of PDAC via miR-203a-3p/MyD88/NF-κB pathway. Data are expressed as the mean ± SD. * p < 0.05 and *** p < 0.001

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: circCUL2 induces an inflammatory CAF phenotype in pancreatic ductal adenocarcinoma via the activation of the MyD88-dependent NF-κB signaling pathway

doi: 10.1186/s13046-021-02237-6

Figure Lengend Snippet: Clinical implication of circCUL2/miR-203a-3p/IL6 axis in PDAC. A , F , K The relative expression of miR-203a-3p ( A ), MyD88 ( F ) and IL6 ( G ) in 161 PDAC tissues compared to paired NAT. B - C , G - H , L - M Association analysis between miR-203a-3p ( B - C ), MyD88 ( G - H ) and IL6 ( L - M ) expression levels and LN status and tumor stages in 161 PDAC tissues. ( D - E , I - J , N - O ) Kaplan-Meier analysis of the correlation between miR-203a-3p ( D - E ), MyD88 ( I - J ) and IL6 ( N - O ) expression levels and OS or DFS of 161 PDAC patients. The median miR-203a-3p, MyD88 and IL6 expression was used as the cutoff value. P Proposed model indicates the mechanism by which circCUL2 activated iCAF phenotype and production of IL6 to promote malignant progression of PDAC via miR-203a-3p/MyD88/NF-κB pathway. Data are expressed as the mean ± SD. * p < 0.05 and *** p < 0.001

Techniques: Expressing

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