Journal: bioRxiv

Article Title: Co-activation of NF-κB and MYC renders cancer cells addicted to IL6 for survival and phenotypic stability

doi: 10.1101/2020.04.12.038414

Figure Lengend Snippet: (A) Enrichment for genes in the signature “Hallmark_Apoptosis” using GSEA and the GEP of spleen CD19 low CD138 + Plasma-cells from C57BL6 mice and C-IM cancer cells. (B) Pathway analysis enrichment (Metacore) of the GEP of C-IM cancer cells compared to spleen CD19 low CD138 + Plasma-cells. The top 5 enriched pathways are shown. (C) RNAseq expression data for genes related to the IL-6 signaling pathway, Socs3 and Il6st . GCB: Germinal Center B-cells, B1: B1 B-cells, FOB: Follicular B-cells, MZB: Marginal Zone B-cells, SPLPC: Spleen Plasma-cells, BMPC: Bone Marrow Plasma-cells, SPLPB: Spleen Plasmablasts , C-IM : GFP + hCD2 + C-IM cancer cells (FACS purified). (D) Representative flow cytometric analysis of the proliferation profiles illustrating the frequency of (pre)plasmablasts (CD138 + ) on day 2, 3, and 4 of LPS stimulation of B-cells from CD19cre IKK2ca stopFL MYC stopFL (IKK2caMYC) in vitro , in the presence of either control antibody (control Ig) or ant-IL6 neutralizing (anti-IL6). Cell Trace Violet (CTV) dye dilution was used to assess proliferation. (E) MFI of pSTAT3 in B220 low CD138 + (pre)plasmablasts at day 2 of in vitro culture of B-cells from the indicated genotypes. Black circles represent control mice (WT), yellow circles CD19cre IKK2ca stopFL mice (IKK2), blue circles CD19cre MYC stopFL (MYC), and red circles CD19cre IKK2ca stopFL MYC stopFL (IKK2MYC). White background: control Ig, dashed background: anti-IL6. (F) Fold change (anti-IL6/control Ig) of the fraction of cleaved caspase 3 + cells within B220 low CD138 + (pre)plasmablasts at day 2 of in vitro culture of B-cells from the indicated genotypes as in (E). (G) MFI of BCL-xL in B220 low CD138 + (pre)plasmablasts at day 2 of in vitro culture of B-cells from the indicated genotypes as in (E). White background: control Ig, dashed background: anti-IL6. (H) MFI of MCL-1 in B220 low CD138 + (pre)plasmablasts at day 2 of in vitro culture of B-cells from the indicated genotypes as in (E). White background: control Ig, dashed background: anti-IL6. (I) Principal component (PC) analysis plots of RNA-seq analysis of activated B cells (B220 + ) and B220 low CD138 + (pre)plasmablasts at day 3 of in vitro culture of B-cells from CD19cre IKK2ca stopFL MYC stopFL (IKK2.MYC). (J) Enrichment for genes in the signature “Hallmark_Apoptosis” using GSEA and the GEP of B220 low CD138 + (pre)plasmablasts at day 3 of in vitro culture of B-cells from CD19cre IKK2ca stopFL MYC stopFL in the presence of control Ig (IKK2.MYC control Ig) or ant-IL6 antibody (IKK2.MYC anti IL6). (K) Cancer free survival curve for C-IM mice treated with control Ig (solid red line) or ant-IL6 antibody (dashed red line).

Article Snippet: To study the impact of IL6 on cancer progression, C-IM mice were treated with InVivoMAb anti-mouse IL-6 (bioXcell), or a control antibody (InVivoMAb rat IgG1 isotype control, anti-horseradish peroxidase; bioXcell).

Techniques: Expressing, Purification, In Vitro, RNA Sequencing Assay

Journal:

Article Title: Transcriptome Analysis of Murine Macrophages in Response to Infection with Streptococcus pyogenes Reveals an Unusual Activation Program ▿

doi: 10.1128/IAI.00181-07

Figure Lengend Snippet: Genes differentially expressed in resident macrophages at 1 h postinfection with S. pyogenes

Article Snippet: In brief, 96-well microtiter plates were coated overnight at 4°C with purified rat anti-mouse anti-IL-6 capture antibody (Pharmingen, San Diego, CA) at 2 μg/ml in sodium bicarbonate buffer.

Techniques: Zinc-Fingers, Binding Assay

Journal:

Article Title: Transcriptome Analysis of Murine Macrophages in Response to Infection with Streptococcus pyogenes Reveals an Unusual Activation Program ▿

doi: 10.1128/IAI.00181-07

Figure Lengend Snippet: Confirmation of microarray data by RT-PCR and protein expression. (A) RT-PCR analysis of selected gene transcription in resident macrophages uninfected or infected with S. pyogenes. Uninfected samples were loaded in lanes 1, and infected samples were loaded in lanes 2. β-Actin expression served as a control. (B) IL-6 protein expression by S. pyogenes-infected macrophages. Resident macrophages were isolated from the peritoneal cavity of mice after 1 h of infection with S. pyogenes and cultured in vitro for 2 h. Noninfected macrophages were used as a control. The levels of IL-6 in the supernatants were determined by ELISA. Each column represents the mean ± standard deviation of triplicate samples obtained from three independent experiments. P < 0.0001 (ANOVA).

Article Snippet: In brief, 96-well microtiter plates were coated overnight at 4°C with purified rat anti-mouse anti-IL-6 capture antibody (Pharmingen, San Diego, CA) at 2 μg/ml in sodium bicarbonate buffer.

Techniques: Microarray, Reverse Transcription Polymerase Chain Reaction, Expressing, Infection, Isolation, Cell Culture, In Vitro, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal:

Article Title: Granulocyte-macrophage colony-stimulating factor regulates cytokine production in cultured macrophages through CD14-dependent and -independent mechanisms

doi: 10.1046/j.1365-2567.2000.00117.x

Figure Lengend Snippet: Cytokine production in 3-day-old (a) and 11-day-old (b) macrophages stimulated by LPS or PMA and cultured with or without GM-CSF and M-CSF. The amount of the different cytokines is shown as the mean of three experiments, with three different donors, each carried out in triplicate. Cytokine production has been calculated as follows: pg of cytokine per ml of supernatants/(no. of live cells in the well/1 × 105). The error bars represent the SD. No intracellular sequestration of cytokines was detected in either control or GM-CSF-macrophages by ELISA testing of cells lysed by repeated cycles of freezing and thawing (data not shown).

Article Snippet: After two further washes, the cells were resuspended for 30 min at room temperature in 30 µl PBS containing 0·1% saponin, 1% bovine serum albumin and 0·5 µg/10 6 cells of the following monoclonal antibodies: Phycoerythrin (PE) -conjugated mouse anti-IL-6 (PharMingen) and FITC mouse anti-human TNF-α.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Granulocyte-macrophage colony-stimulating factor regulates cytokine production in cultured macrophages through CD14-dependent and -independent mechanisms

doi: 10.1046/j.1365-2567.2000.00117.x

Figure Lengend Snippet: Levels in supernatants of IL-10 and IL-12 (pg/ml; mean ± SD) in control and GM-CSF macrophages stimulated or not with LPS and PMA

Article Snippet: After two further washes, the cells were resuspended for 30 min at room temperature in 30 µl PBS containing 0·1% saponin, 1% bovine serum albumin and 0·5 µg/10 6 cells of the following monoclonal antibodies: Phycoerythrin (PE) -conjugated mouse anti-IL-6 (PharMingen) and FITC mouse anti-human TNF-α.

Techniques:

Journal:

Article Title: Granulocyte-macrophage colony-stimulating factor regulates cytokine production in cultured macrophages through CD14-dependent and -independent mechanisms

doi: 10.1046/j.1365-2567.2000.00117.x

Figure Lengend Snippet: Effect of GM-CSF on the frequency of LPS-responding macrophages. The figure demonstrates a representative flow cytometric analysis of control and GM-CSF-macrophages stimulated by LPS and analysed for the frequency of intracellular TNF-α and IL-6 staining. The data are displayed as dot plots. The quadrants were set according to the negative controls (less than 1% of the isotype control cells appeared positive). Five thousand cells were gated and analysed for each sample. The data refer to a typical experiment out of three performed with similar results.

Article Snippet: After two further washes, the cells were resuspended for 30 min at room temperature in 30 µl PBS containing 0·1% saponin, 1% bovine serum albumin and 0·5 µg/10 6 cells of the following monoclonal antibodies: Phycoerythrin (PE) -conjugated mouse anti-IL-6 (PharMingen) and FITC mouse anti-human TNF-α.

Techniques: Staining

Journal:

Article Title: Granulocyte-macrophage colony-stimulating factor regulates cytokine production in cultured macrophages through CD14-dependent and -independent mechanisms

doi: 10.1046/j.1365-2567.2000.00117.x

Figure Lengend Snippet: Effect of GM-CSF on the cytokine intensity of fluorescence of LPS-responding cells. The results are presented as the mean ratio ± SD of the cytokine intensity of fluorescence (arbitrary units) observed among cells responding to LPS. Data are calculated on 1000 events additionally gated on the responding fraction (i.e. the positive cells in the dot plots of Fig. 2).

Article Snippet: After two further washes, the cells were resuspended for 30 min at room temperature in 30 µl PBS containing 0·1% saponin, 1% bovine serum albumin and 0·5 µg/10 6 cells of the following monoclonal antibodies: Phycoerythrin (PE) -conjugated mouse anti-IL-6 (PharMingen) and FITC mouse anti-human TNF-α.

Techniques: Fluorescence