Figure 3. Ablation of RNase L activity attenuates pIC induced cell death and accelerates cell migration.

(a) WT and RNase L, MAVS, PKR KO and RNASEL-MAVS DKO A549 cells were treated with pIC (0 to 5 μg/ml as indicated) and 48 hr later fixed and stained with crystal violet. Data are from one representative experiment of three. (b) WT and KO A549 cells as indicated were transfected with 20 ng/ml pIC and assessed for dead cells and total cells using an IncuCyte system calculated as describe in Methods. Four separate wells were treated with each experimental condition and a minimum of 4 image fields (>10000 cells per well) were analyzed per well. (c) Cells were transfected with 20 ng/ml of pIC and caspase-3/7 activity was determined using an IncuCyte system. Four separate wells were treated with each experimental condition and a minimum of 4 image fields (>10000 cells per well) were analyzed per well. Data represent the means±SD from a minimum of four independent replicates. Similar data were obtained from two additional independent experiments. (d) Cell monolayers were scratched and wound healing was assayed in the IncuCyte system. Wound closure was observed every hour at the indicated times by comparing the mean relative wound density of at least eight technical replicates and a minimum of 2 image fields (>1000 cells per well total) were analyzed per well. Error bars represent standard deviation (SD) from the mean of a minimum of eight. Similar data were obtained from two independent experiments. (e) WT or RNASEL KO HME cells were transfected with 0(mock) or 20, 50, 100 or 250 ng/ml pIC and cell viability was quantified in the IncuCyte system. Four biological replicate wells were treated with each experimental condition and a minimum of 4 image fields (>1000 cells per well total) were analyzed per well. Data is representative of four replicates.

DOI: http://dx.doi.org/10.7554/eLife.25687.008