Inoculation with PrPSc
For use as an inoculum, mechanical lysates of the Rov9Sc (Rov9 cells infected with PrPSc) and Rov9C (Rov9 cells not infected with PrPSc) cells were prepared as previously described [46]. Briefly, the Rov9 cells were grown to confluence in two 75-cm2 plastic tissue culture flasks. Rov9Sc and Rov9C cells were rinsed three times with sterile 16 Dulbecco’s-PBS (D-PBS) and scraped into 10 ml of PBS. The cell pellets were collected by centrifugation at 2206g at room temperature for 7 min and resuspended in 0.5 ml of filtersterilized 5% glucose. The cell suspensions were frozen and thawed four times and then subjected to 1 to 2 min of sonication in a cup horn sonicator. The inoculum was stored at ?0uC. For inoculation, cells were passed into twelve-well plates and allowed to grow to approximately 60% confluence. Cells were rinsed once with PBS and then overlaid with 200 ml of a 1/20 dilution of either the Rov9Sc lysate (microgliaSc) or the Rov9C lysate (microgliaC) in OPTI-MEM. MicrogliaSc (or Rov9Sc) and microgliaC (or Rov9C) were incubated for 6 hours, and then 200 ml of OMEM (or OMEM-Doxy) was added to each well.
Figure 1. Structure of 2-(2-benzimidazolyl)-5-[4-(2-imidazolino)phenyl]furan dihydrochloride (DB772).were expanded into 25-cm2 tissue culture flasks. Cells were fed every 3 or 4 days with appropriate medium as necessary and serially passaged 1/5 after reaching confluence.
Total protein measurement
Since cells were collected based on a fraction of the cell suspension, total protein was analyzed to normalize samples for the various ELISAs. Aliquots of the cell lysates were diluted 1/5 into lysis buffer and then assayed for total protein using the bicinchoninic acid (BCA) protein assay reagent (Thermo Fisher Scientific Inc, Rockford, IL, USA) following manufacturer’s directions for the microplate-based assay.
DB772 inhibits PrPSc accumulation and prion infectivity
To eliminate BVDV from cultures 2-(2-benzimidazolyl)-5-[4-(2imidazolino)phenyl]furan dihydrochloride (DB772) was used at 4 mM, as this is the concentration that cured primary bovine fibroblasts of pestivirus infection [56]. A 10 mM stock solution of DB772 was prepared in sterile water and stored at ?0uC until use. Rov9Sc, Rov9C, microgliaSc, and microgliaC were each split and cultured separately. There were 8 final treatment groups (Rov9Sc/ DB772 , Rov9Sc/UnTx, Rov9C/DB772, Rov9C/UnTx, microgliaSc/ DB772 , microglia Sc/UnTx, microgliaC/DB772, and microgliaC/UnTx [UnTx stands for untreated]). DB772 was maintained in the appropriate culture medium for four passages. Cells were then grown for an additional four passages without DB772 in the culture medium. Cells were collected after four passages with DB772 (P-4, “end of DB772 Tx”) and after four additional passages without DB772 (P-8, “end of clearance”) for pestivirus quantification, PrPSc quantification, and PrPC quantification (see below). Mechanical lysates of Rov9Sc/DB772 were also collected for prion inoculum creation (see above) to verify that prion infectivity was also inhibited by DB772.
Pestivirus measurement
Pestivirus infection in cells was confirmed by RT-PCR and enzyme-linked immunosorbent assay (ELISA). To detect pestiviral RNA, 50 ng of total RNA was reverse transcribed into cDNA using random hexamers (SuperScript III First-Strand Synthesis System, Invitrogen, Carlsbad, CA, USA). The resulting cDNA was amplified using previous methods [60]. Briefly, DNA was amplified in a reaction consisting of 1 ml of cDNA template, 2 mM of each primer (Table 1), 12.5 ml of JumpStartTM REDTaqH ReadyMixTM Reaction Mix (Sigma-Aldrich, St. Louis, MO, USA) and nuclease-free water to a final volume of 25 ml. The reaction was incubated in a thermocycler (C1000, Bio-Rad, Hercules, CA, USA) under the following conditions: 95uC/5 min; 35 cycles of 95uC/30 sec, 56uC/30sec, 72uC/30 sec; and 72uC/5 min. Isolation of detectable amounts of RNA was confirmed by amplifying glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA using intron-spanning primers (Table 1) [61] under the same PCR conditions described above. Amplification products were separated by agarose gel electrophoresis and visualized by staining with ethidium bromide. BVDV-free bovine turbinate cells were cultured in parallel with the Rov9 and microglial cells and served as negative controls. BVDV antigen was detected by commercial ELISA (HerdChek*, BVDV Antigen Test Kit, IDEXX, Westbrook, ME, USA) following the manufacturer’s directions. Aliquots of cell lysates were diluted appropriately in lysis buffer to normalize protein concentrations (based on BCA results) and then added to the kit ELISA plate. The proprietary ELISA positive and negative controls were used per the manufacturer’s directions. Corrected optical densities were transformed to sample/positive ratio (S/P) using manufacturer’s directions: (sample-negative)/(positive-negative). A standard curve prepared from a half-log dilution series of BVDV-infected cell lysate was used to transform the S/P results to relative concentration of BVDV antigen. Untreated, prioninoculated lysates at passage 4 were set at log 1, and all other results were normalized to this value. The relative amount of BVDV antigen within each passage was compared based on DB772 treatment status and PrPSc inoculation status using a twoway ANOVA followed by the Holm-Sidak method of multiple comparisons with a cutoff P value of 0.05 (SigmaPlot 11.2.0.5).
DB772 concentration-dependence curve
To compare the concentration dependencies of BVDV and PrPSc, microgliaSc and Rov9Sc cells were treated with a dilution series of DB772. Samples were collected and analyzed for BVDV ELISA and PrPSc ELISA as described below. Curcumin (SigmaAldrich, St. Louis, MO, USA), which does not inhibit PrPSc accumulation in Rov9Sc cells [47], was used as a negative control for non-specific cell death-induced PrPSc inhibition. Three independent experiments were analyzed. The 50% tissue culture effective concentration (TCEC50) was determined by nonlinear regression using a four-parameter logistic model for microgliaSc and a two-parameter exponential decay model for RovSc (SigmaPlot ver. 11).
