Cells were seeded onto 8-well µ-slides (ibidi USA, Inc.), then infected at an MOI of 10 PFU/cell and incubated at 37°C for the times indicated in the figure legends. The presence of SGs was analyzed either without additional treatment or after induction of SG formation. To induce SGs, cells were treated with 0.75 mM NaAs for 45 min, and then fixed in 4% paraformaldehyde (PFA) in PBS for 20 min at room temperature. Cells were permeabilized with 0.5% Triton X-100 in PBS, blocked with 5 % BSA, and stained with antibodies specific to G3BP1 (gift from Dr. Richard Lloyd), eIF3b (sc-16377, Santa Cruz Biotechnology, Inc.) and TIAR (8509, Cell Signaling Technology), and corresponding fluorescent secondary antibodies. Images were acquired on a Zeiss LSM800 confocal microscope with a 63X 1.4NA PlanApochromat oil objective.
To analyze the effect of ActD-induced transcriptional shutoff on the ability of cells to generate SGs, NIH 3T3 cells in 8-well µ-slides (ibidi USA, Inc.), were incubated in complete media supplemented with ActD (5 µg/ml) for 1, 2, and 4 h. Then, they were treated with NaAs and stained with TIAR- and eIF3b-specific antibodies as described above.
To analyze the effect of ActD-induced transcriptional shutoff on the ability of cells to generate SGs, NIH 3T3 cells in 8-well µ-slides (ibidi USA, Inc.), were incubated in complete media supplemented with ActD (5 µg/ml) for 1, 2, and 4 h. Then, they were treated with NaAs and stained with TIAR- and eIF3b-specific antibodies as described above.