F6627

PTX (T7402, Millipore Sigma, Burlington, MA, USA), 5-FU (F6627, Millipore Sigma), CPT (22-515-0, Thermo Fisher Scientific) and TMS (10038, Cayman Chemicals, Ann Arbor, Michigan, USA) were dissolved in dimethyl sulfoxide (DMSO; D2650, Millipore Sigma). The final concentration of DMSO in the media did not exceed 0.1%.

Determination of concentration for each anticancer drug in our experiments was based on its reported concentration in human cerebral spinal fluid (CSF) in chemotherapy, literature report of its neurotoxicity in culture, and our preliminary in vitro experimental data. At 13 d.i.v., the neuronal cell cultures were insulted with 100 μM of MTX (M9929, Sigma) [27 (link)–30 (link)], 25 μM of 5-FU (F6627, Sigma) [31 (link)–33 (link)], or 3.5 μM of CDDP (sc-200896, Santa Cruz) [34 (link), 35 (link)] for 3 days in absence or presence of PAN-811·Cl·H₂O. For ROS examination, the neurons were insulted with both 100 μM of MTX and 25 μM of 5-FU by 15 d.i.v. PAN-811·Cl·H₂O was added to cultures to final concentrations of 1.25, 2.5, 5, and 10 μM at the same time as addition of the anticancer drugs. For cancer cell lines, the cells were insulted by the second day of cell seeding with the same concentration of MTX, 5-FU, or CDDP as used in neuronal culture in the absence or presence of 10 μM PAN-811·Cl·H₂O for another 3 days.

Control cells or MAGE-expressing cells were treated with indicated concentrations of 5-Fluorouracil (5-FU, Sigma Aldrich F6627) or Sodium Arsenite (Sigma Aldrich, S7400) for 72 h and viability was measured using Cell-titer Glo (Promega G7570) on a Tecan luminescence plate reader. Viability at each time point was normalized to luminescence of DMSO control. Error bars indicate mean and standard deviation from n = 3 measurements.

HuH7 cells seeded in 6-well clusters were infected with recombinant MERS-CoV at low MOI (30 PFU/well) for 1 h at 37°C. Subsequently, the inoculum was replaced with 2 ml of a 1.2% suspension of Avicel (RC-581; FMC Biopolymer) (101 (link)) in Dulbecco’s minimal essential medium (DMEM) (containing 2% FCS and antibiotics) and serial dilutions of 5-FU (F6627; Sigma-Aldrich) or ribavirin (R9644; Sigma-Aldrich) ranging from 0 to 400 μM. Cells were incubated at 37°C for 72 h and fixed with 7.4% formaldehyde, and plaques were visualized using crystal violet staining.
To compare the effect of 5-FU treatment on the progeny titers of wt and nsp14-E191D rMERS-CoV, confluent monolayers of HuH7 were incubated for 30 min at 37°C with solvent or a range of 5-FU concentrations. The drug was then removed and cells were infected at an MOI of 0.1 for 1 h at 37°C. After removal of the inoculum, EMEM containing 2% FCS and solvent or a matching concentration of 5-FU was added to the wells. Supernatants were collected after 30 h, and rMERS-CoV progeny titers were determined by plaque assay. All drug-treated samples were normalized to the untreated vehicle control, and values were expressed as fold change compared to untreated-virus titers.