Stattic hy 13818

Recombinant murine CXCL10 (250-16), CXCL13 (250-24), G-CSF (250-05), and GM-CSF (315-03) were purchased from PeproTech (Rocky Hill, NJ, USA). Directly conjugated anti-mouse mAbs CD11b-FITC (M1/70), CD45-PE/Cy7 (30-F11), Ly6G-APC/Cy7 (1A8), Ly6C-PE (AL-21) were from BD Pharmingen (San Jose, CA, USA). Carboxyfluorescein succinimidyl ester (CFSE) was from Molecular Probes (Eugene, OR, USA). The Annexin-V-APC apoptosis analysis kit (AO2001-11P-G) was obtained from Sungene (Tianjin, China). The mouse cytokine array panel A (ARY006) was purchased from R&D Systems (Minneapolis, MN, USA). The MDSC isolation kit was from Miltenyi Biotec (Bergisch Gladbach, Germany) and the streptavidin particles were from BD IMag (San Jose, CA, USA). Anti-p-Akt (Ser473, #4060), anti-Akt (#4691), anti-p-mTOR (Ser2448, #5536), anti-mTOR (#2983), anti-p-STAT3 (Tyr705, #9145) Abs were purchased from Cell Signal Technology (Danvers, MA). Anti-STAT3 (F-2) Abs was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rapamycin (HY-10219) and Stattic (HY-13818) were purchased from Med Chem Express (New Jersey, USA).

To test the effects of B7-H4 expression on human dDCs during T. gondii infection, approximately 1.5 × 10⁶ purified human dDCs were divided equally into three groups: uninfected, infected, and B7-H4-neutralized infected groups. In the B7-H4-neutralized infected group, dDCs were preincubated with anti-B7-H4 monoclonal antibody (mAb; 10 μg/ml) (eBioscience, San Diego, CA, USA) for 2 h. Toxoplasma gondii tachyzoites were added to the infected group and the B7-H4-neutralized infected group at a ratio of 2:1 (T. gondii: cells) followed by incubation for 24 h.
To test the role of STAT3 on enzyme and cytokine expression in dDCs during T. gondii infection, purified human dDCs were equally divided into four groups: uninfected, infected, STAT3-inhibitor, and STAT3-inhibitor infected groups. Cells of the STAT3-inhibitor and the STAT3-inhibitor infected groups were preincubated with Stattic (HY-13818, MedChem Express), a STAT3 inhibitor, which inhibited STAT3 phosphorylation. After 2 h, T. gondii tachyzoites were added to the STAT3-inhibitor infected group and the infected group at the same time as described above.

rhG-CSF protein and TGF-β1 peptide were purchased from Peprotech and R&D Systems, respectively. The Stat3 inhibitor Stattic (HY-13818) was obtained from Med Chem Express (MCE). G-CSF-neutralizing antibody (ab9691) was purchased from Abcam. Anti-Stat3 (D1B2J), anti-Stat3 (p-Tyr705) (D3A7), anti-vimentin (D21H3), and anti-N-cadherin (D4R1H) antibodies were obtained from Cell Signaling Technology. Anti-β-actin (60008-1-Ig), anti-CD44 (ab216647), and anti-CD90 (66766-1-Ig) antibodies were from Proteintech. The nonspecific (NS) and two Stat3-specific siRNAs, targeting GGCGTCCAGTTCACTACTA and AGACCCGTCAACAAATTAA, respectively, were purchased from RiboBio.