Okadaic acid

To investigate the role of PP2A in PRRSV infection, we used okadaic acid (ApexBio) as a PP2A inhibitor [48 (link)]. The cytotoxicity effect of okadaic acid was determined by a cell counting kit-8 (CCK-8) system (Dojindo Laboratory, Kumamoto, Japan) according to the manufacturer’s instructions. In brief, PAMs and Marc-145 cells were cultured in 96-well-plate and incubated with carrier control ethanol or okadaic acid at different concentrations (0, 10, 20, and 40 nM) for 24 h. CCK-8 solution (10 μL per 100 μL of medium in each well) was added, and the plates were then incubated at 37 °C for 3 h. The absorbance of each well was read at 450 nm under a microplate reader. Concentrations of <40 nM were nontoxic (see Results), and cells were therefore incubated with 10 or 20 nM for 1 h before they were inoculated with PRRSV. Unbound virus was removed, and cells were further cultured in the presence of the inhibitor for 24 h to assess the effect of drugs on virus replication.
To confirm PP2A function, PAMs and Marc-145 cells were transfected with 100 nM control siRNA and PP2Ac specific siRNA duplexes (Table 2) using Lipofectamine RNAiMAX reagent (Invitrogen, USA) according to the manufacturer’s instructions. At 24 h post transfection, cells were infected with PRRSV at an MOI of 0.1 or 1.0.

The human cell lines HEL (expressing JAK2^V617F), SET-2 (expressing both JAK2^V617F and JAK2^WT) and HL-60 (expressing JAK2^WT) were purchased from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). Cells were grown in RPMI 1640 medium with Glutamax (Gibco, Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Gibco).
Ruxolitinib and LB-100 were purchased from Selleck Chemicals (Houston, TX, USA). These drugs were used at a concentration of 1 µM and 2.5 µM, respectively, apart from in clonogenic assays where they were used at 250 nM and 1 µM. Bafilomycin (25 nM) was purchased from Invivogen (Toulouse, France). Chloroquine and Lys05 were purchased from Sigma-Aldrich (Saint-Louis, MO, USA). Chloroquine was used at 20 µM in cell lines, 10 µM in primary cultures and at 1 µM for clonogenic assays. Lys05 was used in vitro at a concentration of 5 µM, except for in clonogenic assays, in which it was used at 1 µM. SAR405 (3 µM) and Okadaic acid (OA, 5 nM) were purchased from APExBIO (Houston, TX, USA) and Cell Signaling Technology (Danvers, MA, USA), respectively.