Journal: International Immunopharmacology

Article Title: IL-35 interferes with splenic T cells in a clinical and experimental model of acute respiratory distress syndrome

doi: 10.1016/j.intimp.2018.12.024

Figure Lengend Snippet: (A, E) Pretreatment with IL-35 neutralizing antibodies (anti-P35 or anti-Ebi3) aggravated LPS- and CLP-induced ARDS. Lungs from each experimental group were processed for histological examination after H&E staining. Compared with the LPS + IgG group, thickened alveolar wall, alveolar hemorrhage and collapse, inflammatory cells in filtration were more severe and pretreated with IL-35 neutralizing antibodies prior to LPS challenge. (B, D) Lung injury scores were estimated by the method of Mikawa, which is from the following four indicators of lung injury score: alveolar congestion; bleeding; gap or vascular wall neutrophil infiltration or aggregation; alveolar septal thickening or transparent membrane formation. 0 marks: no or very slight damage, 1 marks: mild injury, 2 marks: moderate injury, 3 marks: severe injury, 4 marks: very severe damage, the cumulative increase in the number of lesions of the total score is the pathological score of the ARDS. Pretreatment with IL-35 neutralizing antibodies lungs injury were more severe than LPS + IgG group. **p < 0.01, ***p < 0.001, by the two-way ANOVA followed by LSD multiple comparisons test, compared with the LPS + IgG group.

Article Snippet: Neutralization assays were performed by giving 50 μg anti-mouse IL-35 EBI3 (Rockland Immunochemicals) or anti-mouse IL-12A p35 (Abcam Systems) 30 min after CLP or LPS.

Techniques: Staining, Filtration, Membrane

Journal: International Immunopharmacology

Article Title: IL-35 interferes with splenic T cells in a clinical and experimental model of acute respiratory distress syndrome

doi: 10.1016/j.intimp.2018.12.024

Figure Lengend Snippet: Interleukin 35 (IL-35) blockade upregulated the production of proinflammatory cytokines and downregulated anti-inflammatory cytokines during ARDS models. Cytokine and chemokine concentrations in BALF and blood specimens from five mice treated with or without anti–Ebi3 or anti–P35 blocking antibodies were determined by mice cytokine/chemokine magnetic bead panel kit assays 24 h after onset of ARDS. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, by the one-way ANOVA followed by LSD multiple comparisons test, compared with mice treated with isotypical IgG control. ARDS featured increased CXCL-1, TNF-α, IL-6, and IL-17A and reduced IFN-γ, IL-10, IL-2, and IL-13 after neutralizing antibody treatment (anti-IL-35 Ebi3 or anti-IL-35 P35) in BALF and serum.

Article Snippet: Neutralization assays were performed by giving 50 μg anti-mouse IL-35 EBI3 (Rockland Immunochemicals) or anti-mouse IL-12A p35 (Abcam Systems) 30 min after CLP or LPS.

Techniques: Blocking Assay, Control

Journal: Cells

Article Title: Exploring the Interplay of RUNX2 and CXCR4 in Melanoma Progression

doi: 10.3390/cells13050408

Figure Lengend Snippet: Impact of RUNX2 expression on autophagy markers in melanoma cells. ( a ) Western blots of 1A/1B-light chain 3 (LC3) and p62 in MELHO and in the 1E7, 1B3, and 1F5 RUNX2 KO melanoma cell lines. ( b ) Western blots ( left ) and densitometry analyses ( right ) of LC3 and p62 protein levels in MELHO and in the 1F5 RUNX2 KO cell line mock-treated or treated with bafilomycin A1 or 3-methyladenine. ( c ) Western blots ( left ) and densitometry analyses ( right ) of RUNX2 and LC3 in the 1F5 cell line, in the absence or presence of transfection with the pcDNA3/RUNX2 plasmid at 1.3 µg/mL (RUNX2 ++) or 2.6 µg/mL (RUNX2 +++). ( d ) Western blots ( left ) and densitometry analyses ( right ) of beclin in MELHO and in 1F5 cells without or with transfection with the pcDNA3/RUNX2 plasmid. β-Actin was used as a loading control. Western blot images are representative of three independent experiments. Asterisks indicate statistically significant differences relative to the control (( b , c ), right ) or 1F5 RUNX2 KO melanoma cells ((( d ), right ); *: p < 0.05; **: p < 0.005; ***: p < 0.001). Original blots are presented in .

Article Snippet: The PVDF membranes were probed with specific primary antibodies, to detect RUNX2 (1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA, reference: 8486), CXCR4 (1:100 dilution; Abcam, Cambridge, UK, reference: ab124824), MMP13 (1:500 dilution; GeneTex, Irvine, CA, USA, reference: GTX 100665), RANKL (1:500 dilution; Santa Cruz Biotechnology, Dallas, TX, USA, reference: sc377079), LC3B (1:1000 dilution; Invitrogen, Waltham, MA, USA, reference: PA5-22939), p62/SQSTM1 (1:1000 dilution; Rockland, Baltimore, MD, USA, reference: 600-401-HB8), beclin 1 (1:1000 dilution; GeneTex, Irvine, CA, USA, reference: GTX133555), mTOR (1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA, reference: 2983), P-mTOR (1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA, reference: 5536), p70-S6 (1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA, reference: 9202), P-p70-S6 (1:500 dilution; Cell Signaling Technology, Danvers, MA, USA, reference: 9205), and β-actin (1:10,000 dilution; Thermo Scientific, Waltham, MA, USA, reference: BA3R).

Techniques: Expressing, Western Blot, Transfection, Plasmid Preparation, Control

Journal: European journal of immunology

Article Title: Human tolerogenic dendritic cells produce IL-35 in the absence of other IL-12 family members

doi: 10.1002/eji.201445217

Figure Lengend Snippet: (A) DCs and tolDCs were harvested on day 6, replated and stimulated with IFN-γ+LPS;. (B) Cells were stimulated with IFN-γ+LPS, IFN-γ+CD40L, LPS+CD40L or CD40L for 48 hours. (A and B) IL-27 levels were assessed by ELISA. Data are shown as mean ± SD (n=2 replicates) and are pooled from 3-6 independent experiments. (C) tolDCs were cultured on 8 well chamber slides and either untreated, with IFN-γ or IFN-γ+LPS followed by overnight incubation with Brefeldin A. The cells were then fixed and permeabilised, followed by incubation with purified anti-huEbi3 or IL-12p35-allophycocyanin. Ebi3 was detected using GaM-Alexa488.

Article Snippet: Gels were transferred to nitrocellulose membranes, blocked with TBS plus 0.1% Tween-20 (TBS-T) and 3% BSA or milk and probed with indicated primary antibodies; purified anti-IL-12p40, anti-IL-12p35 (Santa Cruz Heidelberg, Germany) or purified anti-Ebi3 (Abnova, Huissen The Netherlands).

Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture, Incubation, Purification

Journal: European journal of immunology

Article Title: Human tolerogenic dendritic cells produce IL-35 in the absence of other IL-12 family members

doi: 10.1002/eji.201445217

Figure Lengend Snippet: Real Time PCR Oligonucleotide sequences

Article Snippet: Gels were transferred to nitrocellulose membranes, blocked with TBS plus 0.1% Tween-20 (TBS-T) and 3% BSA or milk and probed with indicated primary antibodies; purified anti-IL-12p40, anti-IL-12p35 (Santa Cruz Heidelberg, Germany) or purified anti-Ebi3 (Abnova, Huissen The Netherlands).

Techniques: Real-time Polymerase Chain Reaction, Sequencing

Journal: International Journal of Molecular Sciences

Article Title: Impaired Regulation by IL-35 in Systemic Sclerosis

doi: 10.3390/ijms241310567

Figure Lengend Snippet: Gating strategy for the isolated PBMCs. ( A ) Lymphocytes and monocytes were selected based on their morphology using forward- versus side-scatter (FSC-SSC) dotplots. ( B ) Cells were gated with LIVE/DEAD TM Fixable Violet Dead Cell Stain Kit and FSC. ( C ) Monocytes were gated by CD14 expression and ( D ) the percentage of IL-35R+ was analyzed by combining gp130 and IL-12Rβ2. ( E ) Combining anti-CD4 and anti-CD8, we identified CD8+ and CD4+ T cells. ( F ) Tregs were identified on CD4+T cells by high CD127-CD25 expression. IL-35R was evaluated in each population as shown in D. ( G ) Percentage of intracellular IL-35 was analyzed on CD4+ T cells by combining IL-12p35 and EBI3 antibodies. ( H ) Percentages of intracellular IL-10 and ( I ) IL-17 were analyzed on CD4+ T cells.

Article Snippet: After surface staining, the PBMCs were intracellularly stained with anti-IL-12p35-PE (eBioscience, San Diego, CA, USA), anti-EBI3-APC (BD Biosciences, San Jose, CA, USA), anti-IL-10-PE (Miltenyi Biotech), and anti-IL-17-APC (Miltenyi Biotech) on CD4+ T cells after being fixated and permeabilized with a BD Cytofix/Cytoperm kit (BD Biosciences), according to the manufacturer’s instructions ( ).

Techniques: Isolation, Staining, Expressing