Lps escherichia coli 0111 b4

Recombinant human stress induced Hsp70 (Hsp72) was purchased from Enzo Life Sciences (formerly Assay Designs, and formerly Stressgen Biotechnologies Corporation). We tested conventional, non-LPS-free Hsp70 (NSP-555), which we will refer to as “HE/Hsp70”; and LPS-free Hsp70 (ESP-555), which we will refer to as “Hsp70”. The non-LPS free HE/Hsp70 contains 200 pg of LPS per μg of protein, while the LPS-free Hsp70 contains 1.4 pg of LPS per μg of protein (fewer than 50 IU of LPS), as assessed by the Limulus amebocyte lysate assay in our laboratory. According to the manufacturer, both Hsp70preparations retain their ATPase activity. To heat-denature Hsp70, the protein was boiled for 120 min. Escherichia coli 0111:B4 LPS was from Sigma Chemical Co. (St. Louis, MO, USA); Salmonella typhimurium flagellin and Staphylococcus aureus peptidoglycan were from Invivogen (San Diego, CA, USA), and vaccine-grade Salmonella enterica serovar Typhi porins, with < 200 pg of LPS per μg of protein, were obtained in our laboratory as previously reported [19 (link)].

Macrophage cytotoxicity assays were performed using peritoneal macrophages obtained by lavage, or using MACS-separated CD11b⁺ cells isolated from tumors. IFN-γ/LPS activation of macrophages was performed by 4 h incubation with recombinant mouse IFN-γ (20 U/mL; Peprotech, Rocky Hill, NJ, USA), followed by addition of Escherichia coli 0111:B4 LPS (100 ng/mL; Sigma-Aldrich) and 20 h incubation before being used in further experiments. In some assays, macrophages were fixed by 1 min incubation in 1% formaldehyde or 0.05% glutaraldehyde in PBS on ice and washed extensively. For experiments involving macrophages and Id-specific CD4⁺ T cells, macrophage:T cell ratios of 10:1 were used, based on data from previous reports (16 (link)). Growth inhibition assays were performed by co-culture macrophages and tumor cells, at the indicated ratios, for various amounts of time. Tumor cells were then transferred to new wells, and ³H-thymidine was added for 18 h before harvesting. Growth was expressed as percentage counts per minute (cpm) of tumor cells cultured alone. For transwell assays, macrophages were seeded in 24-well plates, and tumor cells added in 250 µL medium in culture chamber inserts with 0.4 µm pore size (Corning Inc., Corning, NY, USA). Tumor cells were harvested after 24 h and analyzed by flow cytometry as specified below.

The Escherichia coli 0111: B4 LPS, N-hexanoyl-D-sphingosine (C6-ceramide) and zVAD-FMK were obtained from Sigma-Aldrich (St. Louis, MO, USA). DAPK1 inhibitor and ST2825 were purchased from Medchem Express (Monmouth Junction, New Jersey, USA). Recombinant GST-DAPK1 fusion protein was obtained from Millipore (Billerica, MA). Caspase-3 activity detection kit was from Bestbio (Shanghai, China). Quantikine human IL-6 ELISA kit was from R&D Systems (Minneapolis, MN). Annexin V-FITC apoptosis detection kit was ordered from Beyotime (Nanjing, China). The following antibodies with the company and concentration were used for coimmunoprecipitation (co-IP) or western-blotting analyses: anti-Pellino1 (Abcam, 1:500), anti-MyD88 (Cell Signaling Technology, 1:500), anti-caspase-8 (Cell Signaling Technology, 1:500), anti-TRIF (Cell Signaling Technology, 1:1000), anti-RIP1 (BD Biosciences, 1:2000), anti-Flag (ProteinTech group, 1:1000), anti-phospho-DAPK1 (Sigma-Aldrich, 1:1000), anti-DAPK1 (Cell Signaling Technology, 1:1000), anti-Fbxw7 (Abcam, 1:1000), anti-pSer (Santa Cruz, 1:1000), anti-Fn14 (Cell Signaling Technology, 1:2000) and anti-GAPDH (Biosynthesis, 1:3000).