Journal: Oncotarget

Article Title: Novel chemokine-like activities of histones in tumor metastasis

doi: 10.18632/oncotarget.11226

Figure Lengend Snippet: A-C. Histones (50 μg/ml, 24 hours) induced chemokine production and release as demonstrated with a Proteome Profiler™ Antibody Array in Hepa1-6 cells. D. Knockdown of NF-κB p65 and TLR4 (but not TLR2 and RAGE) in Hepa1-6 cells inhibited histone (50 μg/ml, 24 hours)-induced CCL9/10 release as demonstrated by ELISA assay (n=3, *, p<0.05 versus control shRNA group). E. Anti-CCL9/10 neutralizing antibody (1 mg/ml) partly inhibited histone (50 μg/ml, 24 hours)-induced Hepa1-6 cell migration (n=3, *, p<0.05).

Article Snippet: The production or release of chemokines was assayed using a Proteome ProfilerTM Antibody Chemokine Array Kit (#ARY020) from R&D Systems Inc. according to the manufacturer's instructions.

Techniques: Ab Array, Knockdown, Enzyme-linked Immunosorbent Assay, Control, shRNA, Migration

Journal: Oncotarget

Article Title: Novel chemokine-like activities of histones in tumor metastasis

doi: 10.18632/oncotarget.11226

Figure Lengend Snippet: A-B. Compared with the control group, TLR4 depletion (by using TLR4 −/− mice or TLR4 knockdown cells) or inhibition of histone release (by administration of 10 mg/kg heparin or 10 mg/kg H3 neutralizing antibody) limited the formation of lung metastasis (as shown in arrow) in mice based on tail vein injection of 3×10 6 Hepa1-6 cells (N=5 mice/group, *, p<0.05 versus control group). In contrast, control IgG (10 mg/kg) did not inhibit the formation of lung metastasis (B). C. Serum nucleosome levels were reduced after treatment with heparin in wildtype, but not in TLR4 −/− mice (N=5 mice/group, *, p<0.05 versus control group). D. Conceptual relationships between histone and tumor metastasis. Histone is a nuclear DAMP and can be released during cell injury or death. Once released, histone can promote cell migration and invasion through the TLR4-ERK-NF-κB pathway, which induces chemokine production and release.

Article Snippet: The production or release of chemokines was assayed using a Proteome ProfilerTM Antibody Chemokine Array Kit (#ARY020) from R&D Systems Inc. according to the manufacturer's instructions.

Techniques: Control, Knockdown, Inhibition, Injection, Migration

Journal: British Journal of Cancer

Article Title: Antibody targeting of CD24 efficiently retards growth and influences cytokine milieu in experimental carcinomas

doi: 10.1038/bjc.2013.102

Figure Lengend Snippet: Effects of SWA11 mAb treatment on intratumoural cytokine milieu in A549 tumours. ( A ) The levels of 40 different cytokines were determined in tumour lysates using a cytokine protein arrays. The level of cytokines was quantified using ImageJ software. Representative arrays from two independent SWA11 mAb treatment experiments are shown. ( B ) Protein levels of CCL5/RANTES, CXCL9/MIG and CCL2/MCP-1 in tumour lysates ( n =5 per group) were determined by ELISA. Data are normalised to the total protein concentration in tumour lysates. * P <0.05.

Article Snippet: Relative levels of 40 different cytokines and chemokines in xenograft tumour lysates were evaluated using a mouse cytokine antibody array kit (Proteome Profiler, R&D Systems) according to the manufacturer's recommendations.

Techniques: Software, Enzyme-linked Immunosorbent Assay, Protein Concentration

Journal: British Journal of Cancer

Article Title: Antibody targeting of CD24 efficiently retards growth and influences cytokine milieu in experimental carcinomas

doi: 10.1038/bjc.2013.102

Figure Lengend Snippet: Effects of SWA11 mAb treatment on intratumoural cytokine milieu in SKOV3ip tumours. ( A ) The levels of 40 different cytokines were determined in tumour lysates using cytokine protein arrays. The level of cytokines was quantified using ImageJ software. Representative arrays from two independent SWA11 mAb treatment experiments are shown. ( B ) Protein levels of CCL5/RANTES, CXCL9/MIG and CCL2/MCP-1 in tumour lysates ( n =5–6 per group) were determined by ELISA. Data are normalised to the total protein concentration in tumour lysates.

Article Snippet: Relative levels of 40 different cytokines and chemokines in xenograft tumour lysates were evaluated using a mouse cytokine antibody array kit (Proteome Profiler, R&D Systems) according to the manufacturer's recommendations.

Techniques: Software, Enzyme-linked Immunosorbent Assay, Protein Concentration

Journal: British Journal of Cancer

Article Title: Antibody targeting of CD24 efficiently retards growth and influences cytokine milieu in experimental carcinomas

doi: 10.1038/bjc.2013.102

Figure Lengend Snippet: Effects of combined SWA11 mAb and gemcitabine treatment on A549 tumour growth and intratumoural cytokine milieu. ( A ) SCID beige mice with established xenograft A549 lung carcinomas received treatment with SWA11 mAb (10 mg kg −1 ) or IgG 2A (10 mg kg −1 ) followed by gemcitabine at a dose of 12 mg kg −1 (10% of maximal tolerated dose) 1 day later ( n =5 per group). Control animals received SWA11 mAb (10 mg kg −1 ), IgG 2A (10 mg kg −1 ) or gemcitabine (12 mg kg −1 ) alone ( n =5 per group). Treatment was repeated three times with a 5-day interval. External size of A549 tumours was measured with a caliper. * P <0.05. Note that part of the results from this experiment are also shown in for illustration. ( B ) Effects of SWA11 mAb and gemcitabine treatment on the levels of 40 different cytokines were determined in A549 tumour lysates using a cytokine protein arrays. The level of cytokines was quantified using ImageJ software.

Article Snippet: Relative levels of 40 different cytokines and chemokines in xenograft tumour lysates were evaluated using a mouse cytokine antibody array kit (Proteome Profiler, R&D Systems) according to the manufacturer's recommendations.

Techniques: Control, Software

Journal: Advanced Science

Article Title: ERK Inhibition Promotes Engraftment of Allografts by Reprogramming T‐Cell Metabolism

doi: 10.1002/advs.202206768

Figure Lengend Snippet: Antibodies and dyes used for western blotting and flow cytometry

Article Snippet: Mouse Th1/Th2/Th17 CBA Kit , BD Biosciences , cat#:560485; RRID: AB_2869354.

Techniques: Western Blot

Journal: Breast Cancer Research : BCR

Article Title: Granulocytic immune infiltrates are essential for the efficient formation of breast cancer liver metastases

doi: 10.1186/s13058-015-0558-3

Figure Lengend Snippet: 4T1-derived breast cancer populations possess distinct and overlapping chemokine profiles. A heatmap representing the secreted levels of 25 chemokines measured using a chemokine array. The chemokines are grouped based on the immune cell types that they are known to recruit (multiple immune cell types, macrophages and/or lymphocytes, T lymphocytes and/or neutrophils, macrophages, T lymphocytes or neutrophils). Red color indicates chemokines that are highly expressed and the blue color denotes chemokines that are underexpressed.

Article Snippet: To detect and quantify chemokines secreted by each cell population, serum-free conditioned media (CM) was collected from cells cultured for 48 h and applied to a chemokine (mouse) quantitative antibody array (Abnova Corp., Taipai, Taiwan, cat#AA0118) following the manufacturer’s protocol.

Techniques: Derivative Assay

Journal: Microbiology Spectrum

Article Title: Density Analysis of Enterovirus D68 Shows Viral Particles Can Associate with Exosomes

doi: 10.1128/spectrum.02452-21

Figure Lengend Snippet: Immune recognition of various EV-D68 densities and characterization of membrane-associated virus. (A) The y axis represents average dilutions of anti-EV-D68 mouse serum required to neutralize virus, divided by the average TCID 50 for respective viral densities (ANOVA post hoc Student'’s t test, P = 0.42, P = 0.68, P = 0.70). (B) Three viral density isolates (1.11, 1.20, and 1.24 g/cm 3 ) were treated with 0.01 mg/mL 15C5-Chmra antibody for 1 h, then mix was put onto TCID 50 plates to assess the viral titer of each isolate. Gray highlight represents detection limit. Asterisks (*) indicate statistical significance (1.11 g/cm 3 , P = 0.0003; 1.20 g/cm 3 , P < 0.0001; 1.24 g/cm 3 , P = 0.0064), all versus respective control, determined by Dunnett’s Method. (C) 15C5-Chmra antibody bound to magnetic beads was added to membrane-associated and naked virus. After 1 h, a magnet was used to remove antibody and the supernatant was added to a TCID 50 plate to assess viral titer (15C5-Chmra versus control: *, P < 0.0005 for both membrane-associated and naked virus; Dunnett’s Method). (D) ICAM-5 or N -acetylneuraminic acid (sialic acid) were attached to magnetic beads and the antibody/bead complex was incubated with membrane-associated or naked virus samples for 1 h. Beads were rinsed twice in excess PBS and viral titer was assessed to determine how much virus was immunoprecipitated from the supernatant (control versus ICAM5 and control versus sialic acid for membrane-associated and naked virus; *, P = 0.0001 determined by Dunnett’s Method). (E) Exosome antibody array on 1.11 g/cm 3 fraction, examining cytosolic proteins (FLOT1, ALIX, TSG101), transmembrane proteins (CD63, CD81, ANXA5), and cis -golgi matrix protein as markers for cellular contamination (GM130). Example blot is shown on the right and chart represents average intensity across three biological replicates. Positive control indicates detection reagents are working correctly, and do not represent an exosome-specific control. Error bars represent standard deviation. Statistics: comparison with control (blank) using Dunnett’s Method ( P = 0.999 for GM130; *, P = 0.027 for FLOT1; P = 0.218 for ICAM; *, P = 0.005 for ALIX; P = 0.086 for CD81; *, P < 0.0001 for CD63; P = 0.305 for EpCAM; *, P < 0.0001 for ANXA5; *, P = 0.0008 for TSG101). Asterisks indicate statistical significance. (F) Anti-CD81 or anti-CD63 antibodies were attached to magnetic beads and incubated with membrane-associated virus. Supernatant was discarded, and beads were rinsed and treated with 0.01% NP-40 (to dissolve exosomes and release virus from bead) before TCID 50 measurement. CD81 versus control: *, P = 0.0178; CD63 versus control: *, P = 0.0180 as determined by Dunnett’s Method. Gray highlight represents detection limit. (G) RD or SH-SY5Y cells in TCID 50 plate were infected with MO47 with or without exosomes in the medium. The “A549 exosomes added” bar represents exosome-depleted media to which purified A549 exosomes were added. Gray panel represents TCID 50 plates containing SH-SY5Y cells. Each condition represents 3 biological replicates. Error bars represent standard deviation. ANOVA: *, P < 0.05. Green panel represents TCID 50 plates containing RD cells. Each condition represents 4 biological replicates. Error bars represent standard deviation. ANOVA: *, P < 0.05.

Article Snippet: We followed the coupling protocol from the Dynabeads Antibody Coupling Kit (Thermo Fisher, cat no. 14311D) to covalently attach magnetic beads to the following antibodies: anti-CD81 (1D6) monoclonal antibody (Novus Biologicals NB100-65805), anti-CD63 (H5C6) monoclonal antibody (Novus Biologicals NBP2-42225), 15C5-chimeric monoclonal antibody (generous gift from Michael Pauly at ZabBio), and anti-HSV negative control (generous gift from Michael Pauly at ZabBio).

Techniques: Membrane, Virus, Control, Magnetic Beads, Incubation, Immunoprecipitation, Ab Array, Positive Control, Standard Deviation, Comparison, Infection, Purification

Journal: Communications Biology

Article Title: Macrophage migration inhibitory factor is overproduced through EGR1 in TET2 low resting monocytes

doi: 10.1038/s42003-022-03057-w

Figure Lengend Snippet: a , b Cytokine profile arrays of supernatant collected from cord blood CD34 + cells infected with SCR - and TET2- shRNA-GFP lentiviruses, sorted on GFP expression, and induced to differentiate with stem cell factor (SCF), interleukin-3 (IL-3), Fms-related tyrosine kinase 3 ligand (FLT3L) and granulocyte-colony stimulating factor (G-CSF). a Representative cytokine array with supernatants collected at day 10 of differentiation. The rectangle points to MIF detection. b Quantification of MIF signals, normalized to positive controls. Data are mean +/− SEM of three independent experiments. Paired t test: * P < 0.05. c MIF concentrations determined by ELISA in the supernatant of cells induced to differentiate for indicated time. Data are mean +/− SEM of indicated independent experiments (day 5: n = 6; day 7: n = 5; day 8: n = 3; day 10: n = 7). Paired t test: * P < 0.05; *** P < 0.001. d RT-qPCR analysis of MIF mRNA expression in four TET2 -depleted ( TET2 shRNA, gray bars) and control ( SCR shRNA, black bars) human leukemic cell lines. Data are mean +/− SEM of three biological replicates. Unpaired t test: * P < 0.05; *** P < 0.001; **** P < 0.0001. e Immunoblot of SCR or TET2 shRNA infected leukemic cell lines sorted on GFP expression. Lower panels, quantification after actin normalization using Image J software. f MIF concentrations determined by ELISA in the supernatants of kasumi-1 ( n = 5), M07e ( n = 5), UT-7 ( n = 4) and TF-1 ( n = 5) cells transduced 24 h before with SCR (black squares) or TET2 (gray squares) shRNA. Data are mean +/− SEM of indicated biological replicates. Unpaired-t test: * P < 0.05; ** P < 0.01. g MIF concentrations determined by ELISA in the plasma of two Tet2 -deficient models (1–3 months): K427 knock-out model (wt/wt or Tet2 +/+ n = 4, wt/exc or Tet2 +/− n = 5 and exc/exc or Tet2 −/− n = 5); ANO knock-down model (wt/wt or Tet2 + /+ n = 5, wt/LacZ or Tet2 +/− n = 5 and LacZ/LacZ or Tet2 −/− n = 6). Data are mean +/− SEM of indicated biological replicates. Dunnett’s multiple comparison tests using wt/wt as control: * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: CD34 + collected supernatants were analyzed using human cytokine antibody array (panel A; R&D Systems).

Techniques: Infection, shRNA, Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Control, Western Blot, Software, Clinical Proteomics, Knock-Out, Knockdown, Comparison