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: Journal of Clinical Medicine

Article Title: Thrombin Preconditioning of Extracellular Vesicles Derived from Mesenchymal Stem Cells Accelerates Cutaneous Wound Healing by Boosting Their Biogenesis and Enriching Cargo Content

doi: 10.3390/jcm8040533

Figure Lengend Snippet: The EVs contain various cytokines and chemokines following stimulation with different wound mimetic environments. EVs were isolated from MSCs preconditioned with thrombin (40 U), LPS (1 μg,) H 2 O 2 (50 μM), or hypoxia (O 2 10%). ( A ) Representative screening X-ray film blot of Proteome Profiler Human Angiogenesis Array (PPHAA) for protein cargos of EVs (upper panel) and their representative diagram for the relative content of each protein expressed by fold change of blot intensity normalized with that of the naïve group. A gray dot line represents a three-fold increase over that of the naïve group. ( B ) Levels of angiogenin, angiopoietin-1, VEGF, and HGF measured by muliplex ELISA in EVs. Statistical analyses were carried out using a One-way Analysis of Variance (ANOVA). The asterisk (*) indicates p < 0.05 compared with naive EVs, the number sign (#) indicates p < 0.05 compared with the 10% oxygen-treated EV group, the double dagger (‡) indicates p < 0.05 compared to the H 2 O 2 -treated EV group, and the dollar sign ($) indicates p < 0.05 compared with the LPS-treated EV group ( n = 5 per each analysis).

Article Snippet: The relative expression of 55 angiogenesis-related proteins was determined in each lysate using a Proteome ProfilerTM human angiogenesis array kit (R&D Systems, Abingdon, UK) following the manufacturer’s protocol.

Techniques: Isolation, Enzyme-linked Immunosorbent Assay

Journal: Oncotarget

Article Title: PTEN mediates the cross talk between breast and glial cells in brain metastases leading to rapid disease progression.

doi: 10.18632/oncotarget.14047

Figure Lengend Snippet: Figure 7: Effects of glial cell cocultures on cytokine secretion. Control and PTEN overexpressing MDA-MB-231 BR cells were seeded in transwell inserts and cocultured for 48 h with astrocytes (A) or microglia (B). Antibody-spotted membranes were incubated with conditioned media of these cultures and levels of secreted proteins were visualized by HRP-conjugated antibodies. Proteins with a more than 2-fold expression difference are shown by open boxes. Results were verified by qPCR analyses (C). Astrocyte coculture reduced EGF and GM-CSF expression significantly in PTEN overexpressing (PTEN CoA) compared to control cells (CTL CoA, C). Astrocytes express the GM- CSF receptor (CSFR2A) only when cocultured with tumor cells. Microglia coculture reduced VEGFA expression in PTEN overexpressing (PTEN CoMG) compared to control cells (CTL CoMG). Student’s t-test was used to analyze statistical significance. *p < 0.05.

Article Snippet: Cytokine secretion was estimated from CM by Proteome ProfilerTM Antibody Array (Human XL Cytokine Array Kit, ARY022, R&D Systems, Wiesbaden, Germany) according to manufacturer’s instruction.

Techniques: Control, Incubation, Expressing

Journal: Cancers

Article Title: Visfatin Mediates Malignant Behaviors through Adipose-Derived Stem Cells Intermediary in Breast Cancer

doi: 10.3390/cancers12010029

Figure Lengend Snippet: GDF15 play a crucial role in the regulations of breast cancer cells activities by visfatin-treated ADSCs (vADSCs). ( A ) After the three-day co-culture of MDA-MB-231 cells and vADSCs (V50 and V100 group) or untreated ADSCs (Ctrl group), the CM was collected and analyzed by using a cytokine array kit. ( B ) The expression of GDF15 in the co-cultured CM was validated by ELISA represented in a histogram. The ADSCs and MDA-MB-231 cells collected from the co-culture system were extracted for cell lysate to analyze the GDF15 expression by western blotting. ( C ) The migration and invasion of MDA-MB-231 treated with GDF15 at various concentrations for 48 h were evaluated by using a transwell system. ( D ) The indirect co-culture was performed in the presence or absence of the GDF15 neutralizing antibody of for three days. After that, the migration and invasion of the MDA-MB-231 cells collected from the co-culture were evaluated in a transwell system. ( E ) The expression of phosphor-AKT (pAKT) of MDA-MB-231 treated with GDF15 (50 ng/mL) at different time point was detected by western blotting. ( F ) The pAKT was detected in the MDA-MB-231 cells from the co-culture by western blotting. ( G ) After the three-day co-culture in the presence or absence of the wortmannin (400 nM), the MDA-MB-231 cells were collected from the co-culture for performing the migration assay. All experiments were performed in triplicate.

Article Snippet: To explore the cytokine expression profile in the medium of the indirect co-culture of MDA-MB-231 and resistin-pretreated ADSCs, a Proteome profilerTM human XL cytokine array kit (ARY022, R&D Systems) was adopted.

Techniques: Co-Culture Assay, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot, Migration

Journal: International Journal of Molecular Sciences

Article Title: Insight into Cisplatin-Resistance Signaling of W1 Ovarian Cancer Cells Emerges mTOR and HSP27 as Targets for Sensitization Strategies

doi: 10.3390/ijms21239240

Figure Lengend Snippet: ( a ) Data set of a human proteome profiler kinase activity array displaying the phosphorylated/activated form of the components in cisplatin (cDDP)-treated W1 and W1CR cells with and without collagen ( n = 1). ( b ) Overview of the investigated signaling pathway and inhibitory approaches.

Article Snippet: A Proteome ProfilerTM Human Phospho-Kinase Array Kit (R&D Systems) was performed to examine W1 and W1CR cells for changes in intracellular signaling pathways comparing untreated cells with the effects of COL1 binding and cytotoxic stress by cisplatin (EC 50 ).

Techniques: Activity Assay