Heclin

Epinecidin-1 (GFIFHIIKGLFHAGKMIHGLV-NH2; Epi) peptide was purchased from GL Biochem (Shanghai, China). Lipopolysaccharide (LPS) from Escherichia coli O111:B4, Lipoteichoic acid (LTA) from Staphylococcus aureus, MG132 and Heclin were purchased from Sigma. Epi was dissolved in dissolved in normal saline. LPS and LTA were dissolved in distilled water. MG132 and Heclin were dissolved in DMSO.

For cell lines, human GC cell lines, BGC803, MKN45, SGC7901, and MKN28, were cultured in RPMI-1640 medium (GIBCO BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (GIBCO BRL) and 100 mg/ml penicillin-streptomycin. Human GC cell line AGS was cultured in DMEM (GIBCO BRL) supplemented with 10% fetal bovine serum (GIBCO BRL) and 100 mg/ml penicillin-streptomycin. HEK293T cells were cultured in DMEM (GIBCO BRL) supplemented with 10% fetal bovine serum (GIBCO BRL) and 100 mg/ml penicillin-streptomycin. Cells were incubated at 37 °C in 5% CO2. Human GC cell lines BGC803, MKN45, SGC7901, MKN28, and AGS, were purchased from Genechem (Shanghai, China). HEK293T cells were purchased from ATCC (Manassas, VA, USA). For drugs, OSI906 and Doxycycline were purchased from Sellechem catalog #S1091 and #S4163, respectively, and the IC50 data was obtained from the Selleckchem website (Selleckchem.com). Heclin was purchased from Sigma-Aldrich (SML1396).

Ehrlichia chaffeensis (Arkansas strain) was propagated in a human monocytic cell line (THP-1; ATCC, Manassas, VA). THP-1 cells were maintained in RPMI 1640 with HEPES (25 mM) (Invitrogen; Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS)(HyClone, Logan, UT), 5 mM L-glutamine, 1% sodium pyruvate, and 12.5 ml 10% glucose (Sigma; St. Louis, MO) at 37°C in a 5% CO₂ atmosphere. E. chaffeensis infection was maintained by subculturing infected cells with uninfected. HeLa cells (human cervical epithelial; ATCC) for transfection were grown in MEM (Invitrogen) supplemented with 10% FBS (HyClone) and maintained in a 5% CO₂ atmosphere. When stated cells were incubated with 10 mM bortezomib (ApexBio; Houston, TX), PYR41 (Thermo Fisher; Boston, MA), Heclin (Sigma), carfilzomib (ApexBio) or vehicle (DMSO) for 12–24 h before harvesting cells for lysate or luciferase expression measurement.