Curcumin

Cell viability was measured by using a Cell Counting Kit-8 (CCK-8, Dojindo, Japan) according to the manufacturer’s instructions. Briefly, cells were seeded at a density of 0.5 × 10⁴ cells/well in 96-well plates, and incubated in RPMI medium 1640 supplemented without FBS overnight. Curcumin (Selleck, Houston, United States) was dissolved in dimethyl sulfoxide (DMSO). Cells were treated with various concentrations (0, 5, 10, 20, and 40 μM) of Curcumin for 24 h, and appropriate controls were treated with DMSO at the same concentrations. The samples were tested every 24 h for 3 days. CCK-8 solution (10 μl) was added to each well for 2 h and optical density was measured at 450 nm to estimate viable cells.

Chemotherapeutics, 5-FU (S1224), gemcitabine (S1149) and irinotecan (S2217), were purchased from Selleckchem (Houston, TX, USA). Targeted cancer therapy drugs, gefitinib (S1025) and selumetinib (S1008) were purchased from Selleckchem, while lapatinib (11493) was purchased from Cayman Chemical (Ann Harbor, MI, USA). Fulvestrant (S1191, Selleckchem) and metformin (13118, Cayman Chemical) were used as repurposed agents. Natural products, apigenin (S2262), curcumin (S1848), fisetin (S2298), and forskolin (S2449) were purchased from Selleckchem, while (-)- epigallocatechin gallate (70935), procyanidin B2 (19865), resveratrol (70675) and urolithin A (22607) were purchased from Cayman Chemical. All chemicals had more than 98% purity. Stock solutions were prepared according to the manufacturers’ instructions.

O-phosphorylethanolamine (Sigma, USA, #P0503-1G), hyodeoxycholic acid (Sigma, USA, H3878-5G), L-tryptophanamide (Selleck, USA, #S6155), cyclamic acid (Yuanye Biology, China, #S70017-5G), sodium cyclamate (Sigma, USA, #47827), curcumin [65 (link)] (Selleck, USA, #S1848), bevacizumab (Selleck, USA, #A2006). Blank control group (culture medium), negative control (0.1% DMSO group), curcumin (positive control group when studying the effect of differential metabolites on HRPECs), bevacizumab group (positive control group when studying the effect of differential metabolites on HRECs) were set.

Human embryonic kidney HEK293 cells were obtained from American Type Culture Collection (ATCC) and cultured in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 100 IU/ml of penicillin and 100 μg/ml of streptomycin. HEK293-NSP1 stable cell lines were generated from HEK293 cells and cultured in complete DMEM in the presence of puromycin (0.5 μg/ml). Expression of NSP1 was induced by doxycycline (1 μg/ml) treatment for 24 hr. African Green Monkey kidney MA104 cells were obtained from ATCC and cultured in Medium 199 supplemented with 10% FBS, 2 mM L-glutamine, 100 IU/ml of penicillin and 100 μg/ml of streptomycin. Cells were stimulated with TNF-α (10 ng/ml), IL-1β (10 ng/ml), IFN-β (100 U/ml), poly (I:C) LMW/LyoVec (100 ng/ml) for 15 min or 6 hr for either western blot/immunofluorescence or QPCR quantification, respectively. Cells were treated with either small-molecule proteasome inhibitors (10 μM) for 12 hr: MG-132, bortezomib, carfilzomib, VR23, celastrol, curcumin from Selleckchem, lactacystin and epoxomicin from Enzo Life Sciences; or lysosome inhibitors (10 μM) for 12 hr: chloroquine (InvivoGen), concanamycin A (Enzo), bafilomycin A (Sigma). NEDD8 activating enzyme inhibitor MLN4924 (Millipore) was reconstituted in DMSO (stock concentration: 20 mM) and used at the range of 100 nM to 10 μM.