Lipopolysaccharides (lps)

Bone marrow-derived DCs were generated from C57BL/6 mouse bone marrow as described previously (Liu et al. 2014 (link)). Bone marrow was isolated, and red blood cells were lysed with 0.87% ammonium chloride buffer. The bone marrow cells (1 × 10⁶/ml) were suspended in complete RPMI-1640 medium with 20 ng/ml GM-CSF and 10 ng/ml IL-4 (ProSpec, Ness-Ziona, Israel) for 48 h to generate immature DCs (imDCs) and then divided into four groups: the imDC control group, PF-treated imDC group, LPS-treated imDC group and PF + LPS-treated imDC group. PF (50 μg/ml) was used to treat imDCs stimulated with 1 μg/ml LPS (Sigma‑Aldrich, Hamburg, Germany), and 36 h later, the supernatants were collected.

A transwell coculture system was used to investigate MSC paracrine protective effects on endothelial barrier. MSCs with or without overexpression HGF gene were seeded to upper transwell chamber (0.4 μm pore size polyester membrane from Corning, Inc., USA), and PMVECs were seeded to lower chamber (six-well culture plates). We cultured for 1-3 days to allow the growth of a confluent monolayer and then added LPS (100 ng/ml, Sigma, USA) to PMVECs.
Gram-negative bacterial pathogen lipopolysaccharide (LPS, 100 ng/ml, Sigma, USA) was treated with PMVECs to mimic endothelial barrier dysfunction. To determine the roles and mechanisms HGF, recombinant murine HGF (20 ng/ml, ProSpec, Israel) was introduced to LPS-induced PMVECs. Doses of LPS and HGF were applied according to our preliminary experiments. Moreover, PBS was applied as negative control and Akt inhibitor AZD53631 (1 μM, Selleck, USA) or PKC inhibitor enzastaurin (LY317615) (1 μM, Selleck) was applied to inhibit the activation in PMVECs. Phosphatidylinositol 3,4,5-trisphosphate as PI3K facilitates (PtdIns(3,4,5)P3, 25 ng/ml, Sigma) was also used to active mTORC2 signaling [19 (link)].

The amebicidal activity of activated macrophages was determined using a previously described protocol69 (link). Briefly, RAW 264.7 murine macrophages in DMEM were activated by 22 h of incubation with lipopolysaccharide (LPS) (50 ng/ml), INF-γ (300 u/μl, Prospec), and L-arginine (1 mM, Sigma). Activated macrophages (2 × 10⁶/ml) and E. histolytica trophozoites (2 × 10⁴/ml) were co-incubated in DMEM at 37 °C for 6 h. The viability of trophozoites was measured by determining the ability of trophozoites to exclude eosin dye (0.1% final concentration).