Journal: PLoS Pathogens

Article Title: Reprogramming the unfolded protein response for replication by porcine reproductive and respiratory syndrome virus

doi: 10.1371/journal.ppat.1008169

Figure Lengend Snippet: ( A ) Relative abundance of GRP78 mRNA in PRRSV-infected MARC-145 cells at different time points post infection. The level of GRP78 mRNA was normalized against GAPDH and then compared to the uninfected group. ( B ) Western blot analysis of GRP78 protein levels in MARC-145 cells infected with PRRSV at an MOI of 0.1. TG treatment served as a positive control. β-actin served as a loading control, and the viral nucleocapsid protein, N, was used as an infection indicator. The graph shows the levels of GRP78 normalized against β-actin. ( C and D ) The same as (A) and (B), excepts that primary PAMs were used. ( E ) MARC-145 cells were infected with PRRSV strain JXwn06 at an MOI of 0.1, and at 24 hpi, the cells were treated with MG-132 (30 μM) or Bafilomycin A1 (0.5 μM) or Z-VAD-FMK (20 μM) for 2 h, before being collected for Western blot analyses. The graph shows the quantitative analysis of the GRP78 protein levels. ( F ) Decreasing levels of GRP78 in HEK-293FT cells in response to increasing levels of transiently-expressed PRRSV GP2a. ( G ) HEK-293FT cells grown on coverslips in six-well plates were transfected to express GP2a-HA. At 24 h post transfection, they were treated with MG-132 (30 μM), Bafilomycin A1 (0.5 μM), or Z-VAD-FMK (20 μM) for 6 h before being collected for western blot analyses. ( H ) HEK-293FT cells grown on coverslips in six-well plates were transfected to express GP2a-HA, GP2aΔE-HA, or E-HA. At 24 h post transfection, the cells were collected for Western blot analyses. Data information: Statistical analyses were performed by two-tailed Student’s t -test or one-way ANOVA, and asterisks (*) indicate the statistical significance: NS, no significance; *, P < 0.05; **, P < 0.01 ***; P < 0.001. Data are presented as means ± standard deviations (SD) of three independent experiments.

Article Snippet: MARC-145 cells (African green monkey kidney epithelial cells) (ATCC CRL-12231), Vero cells (African green monkey kidney epithelial cells) (ATCC CRL-1586), BHK-21 cells (Baby hamster kidney) (ATCC CRL-12071), and HEK-293FT cells (Human embryonic kidney cells) (Thermo, #R70007) were all cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, #12491015) with 10% fetal bovine serum (FBS) (Gibco, #16140071) and penicillin (50 U/mL) & streptomycin (50 μg/mL) at 37°C in a humidified atmosphere of 5% CO 2 .

Techniques: Infection, Western Blot, Positive Control, Transfection, Two Tailed Test