Stat3 inhibitor stattic

RPMI-1640 medium containing 10% fetal calf serum was routinely used for primary cell cultures. KnockOut Serum Replacement (Thermo Fisher, Waltham, MA, USA) was used for all migration assays involving S1P. Anti-CD4-PE-Cy7 (GK1.5), anti-CD4-FITC (RM4–5), anti-CD8-PE (53-6.7), anti-CD8-PECF594 (53-6.7), anti-CD69 PerCP-Cy5.5 (H1.2F3), anti-CD44-PE (IM7), anti-CD62L-APC (MEL-14), and anti-p-Stat3-Percp cy5 were purchased from BD PharMingen (San Diego, CA, USA). Anti-CCR2-PE-Cy7 (SA203G11), anti-CD45.2-FITC (104), anti-CD45.1-APC (A20), anti-Qa-2-biotin (695H1-9-9), anti-Qa-2-Alexa Fluor 647 (695H1-9-9), anti-Ki67-PE-Cy7, anti-CXCR3-APC, and anti-CXCR6-PE were purchased from BioLegend (San Diego, CA, USA). Anti-CCR2-APC (Catalog # FAB5538A) and anti-S1P1-PE (Catalog # FAB7089P) were purchased from R&D Systems (Minneapolis, MN, USA). Unlabeled antibodies against Akt, p-Akt (Ser473), STAT3, p-STAT3 (Tyr705), FoxO1 (C29H4), and β-actin were purchased from Cell Signaling Technology (Beverly, MA, USA). Recombinant mouse CCL2, CXCL9, and CXCL16 were purchased from R&D Systems (Minneapolis, MN, USA). Stat3 inhibitor Stattic was purchased from Selleck.

Methamphetamine was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). The specific MEK1/2 inhibitor U0126, JNK inhibitor SP600125, p38 inhibitor SB203580 and phosphatidylinositol-3 kinase (PI3K) inhibitor LY294002 were purchased from Calbiochem (San Diego, CA). The NADPH inhibitor apocynin was obtained from Sigma-Aldrich (St. Louis, MO, USA), and the STAT3 inhibitor stattic was ordered from Selleck (Houston, TX, US). The concentrations of these inhibitors were based on a concentration-curve study and our previous reports^{23 (link)}.

PBMC from healthy donors were cultured in the presence of varying concentrations (10, 30, 50% v/v) of AF from OC patients for 24, 48 or 72 hours, and resultant cells were analyzed for the frequency of or ARG1, iNOS, S100A8/A9 (Santa cruz) expression in MDSC by flow cytometry. In some instances, PBMC were cultured with the AF in the presence of neutralizing antibodies against IL-6 and/or IL-10 (Biolegend) or STAT3 inhibitor stattic (Selleck) with isotype antibody or DMSO as controls. In additional settings, PBMC from healthy donors were treated with recombinant human IL-6 and IL-10 (Peprotech) at indicated concentration with or without STAT3 inhibitor stattic for 48 hours.