methyl-β-cyclodextrin (m-βcd  (Millipore)

Journal: PLoS ONE

Article Title: HIV-1 Tat Protein Induces Production of Proinflammatory Cytokines by Human Dendritic Cells and Monocytes/Macrophages through Engagement of TLR4-MD2-CD14 Complex and Activation of NF-κB Pathway

doi: 10.1371/journal.pone.0129425

Figure Lengend Snippet: ( A ) U937, HEK-TLR4-MD2-CD14 or human monocytes were stimulated or not with 10 or 100 nM of GST +/-Tat for the indicated times. Surface expression of TLR4 in unstimulated (black line) or stimulated (pink, blue and red lines) cells was analyzed by flow cytometry using anti-TLR4 or isotype control IgG (tinted), stained with secondary FITC or PE antibodies. Data represent one of three independent experiments. ( B ) U937 were pre-incubated for 1 h with 1 μg/mL of blocking anti-TLR4 or ( C ) HEK TLR4-CD14-MD2 were pretreated with 80 μM of dynasore for 30 minutes before stimulation with GST+/-Tat (100 nM) for the time periods indicated. Cell surface expression of TLR4 was then analyzed as described above. ( D – G ) The integrity of the raft structure is necessary for Tat-induced IL-6 and IL-8: D-E ) Purified human monocytes (10 6 ) were pretreated with increasing amounts of raft disruption drug M-β-CD (10, 60 min) or F-G ) with dynasore for 30 minutes before stimulation with GST-Tat 1–101 (100 nM). After 24 h, IL-6 and IL-8 production in the culture supernatants were quantified by ELISA. The data represent means (pg/ml) and SD (n >3). As controls, cells were stimulated with PBS, DMSO or the highest drug concentration and cytokine production and cytotoxicity (trypan blue) were analyzed. Asterisks represent P values: *, P ˂ 0.05; **, P ˂ 0.01; ***, P ˂ 0.001, ns non significant.

Article Snippet: The raft disrupting drug methyl–β-cyclodextrin (M-βCD) and a GTPase dynamin inhibitor dynasore were purchased from Sigma-Aldrich.

Techniques: Expressing, Flow Cytometry, Staining, Incubation, Blocking Assay, Purification, Enzyme-linked Immunosorbent Assay, Concentration Assay