Nsc 87877

BMS202 (PD-1 inhibitor), SC79 (Akt activator), and NSC87877 (SHP1/2 inhibitor) were purchased from Selleck Chemicals (Houston, TX, USA). Enzyme-linked immunosorbent assay (ELISA) kits for TNF-α, IL-6, and IL-1β were purchased from Elabscience Biotechnology (Wuhan, China). Antibodies against HIF-1α, SHP1, SHP2, phosphorylated Akt (p-Akt), Akt, and β-actin were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against PD-1, CD11c, CD16, CD86, and CD86/PE were from Bioss (Beijing, China). Fluorophore-labeled goat anti-rabbit secondary antibody (Alexa Fluor 594), goat anti-rabbit immunoglobulin horseradish peroxidase (IgG-HRP), bovine serum albumin (BSA), and the bicinchoninic acid (BCA)-based protein assay were purchased from Beyotime (Shanghai, China). The 2-step plus Poly-HRP Anti Mouse/Rabbit IgG Detection System kit was from Solarbio Life Sciences (Beijing, China). TRIzol reagent was purchased from Invitrogen (Carlsbad, CA, USA); PrimeScript™ RT Kit, from Takara (Osaka, Japan); fetal bovine serum (FBS); and Ham’s F-12 K (Kaighn’s) medium, from Gibco (Carlsbad, CA, USA).

BMDMs were obtained as previously described (38 (link)). Briefly, bone marrow cells, isolated from tibias and femurs of C57BL/6 mice, were cultured in complete DMEM medium supplemented with GM-CSF (10 ng/ml, Peprotech). After 4 days, the cells received a fresh complete DMEM medium supplemented with GM-CSF (10 ng/ml) and were incubated for 3 additional days. BMDMs were harvested and then seeded in fresh complete DMEM medium at a density of 2 × 10⁶ cells/ml for experiments. Under certain condition, anti-CD180 Ab (0.2 μg/ml) or IgG2b antibody (0.2 μg/ml) was added to BMDMs before stimulated with R837 (1 μg/ml, Enzo), CpG 1826 (0.5 μM, Invitrogen) or murine IFN-α (1,000 U/ml, Biolegend); to block the activity of SHP-1 and SHP-2 in BMDMs, the cells were pretreated with SHP-1 and SHP-2 inhibitor NSC-87877 (1 μM, SELLECK) for 30 min and then used for subsequent experiments. The vehicle used to delivery anti-CD180 antibody and IgG2b antibody is phosphate-buffered solution containing no preservative.

Human cancer cell lines originated from the CCLE [18 (link)] were authenticated by single-nucleotide polymorphism analysis and tested for mycoplasma infection using a PCR-based detection technology (RADIL, University of Missouri, Columbia, MO; now IDEXX Laboratories). All cell lines used were directly thawed from the CCLE collection stock and cultured as previously described [18 (link)] except SW1736 (Creative Bioarray). Cell lines were used within 15 passages of thawing and continuously cultured for less than 6 months. All small molecule inhibitors used were synthesized and structurally verified by NMR and LC/MS according to cited references at Novartis except RMC-4550 (Selleck Chemicals), NSC-87877 (Selleck Chemicals), and IIB-08 (Millipore Sigma). EGF and FGF19 were purchased from R&D Systems.