Figure 1
From: Suppression of Rac1 Signaling by Influenza A Virus NS1 Facilitates Viral Replication
NS1 modulates cell morphology and migration.
(A) Generation of the NS1 stable cell lines. A549 cells were transfected with the pcDNA3.0-flag or pcDNA3.0-flag-NS1 plasmid. After selection in 800 mg/L G418 for 3 weeks, the NS1 expression levels were detected in the stably transfected cell lines by western blotting using anti-flag antibody. (B) The NS1 expression levels in the NS1-positive cell lines were determined by fluorescence-activated cell sorting (FACS). The indicated cell lines were fixed, permeabilized, and stained for Flag-NS1. ModFit LT version 2.0 was utilized to determine the percentage of NS1-positive cells. (C) Scanning electron microscopy (SEM) showed the morphological differences between the control cell lines (N1, N2, and N3) and the NS1 over-expressing cell lines (P1, P2, and P3). The cell lines were cultured for 24 hours, and then fixed, dried, sprayed with metal and photographed by SEM. White arrows indicate the cellular lamellipodia. (D) The percentage of the cell spreading area. Image J software was used to analyze the cell spreading area. The results are presented as the mean ± SD of triplicate experiments (***p < 0.001 relative to the control group). The data were evaluated using a t-test with the SPSS 11.5 software program (SPSS, Inc., Chicago, IL, USA). (E) A wound healing assay was performed to detect the migration ability in the two different cell lines. The indicated cell lines were seeded into six-well plates. After the cells grew to confluence, wounds were made using 10 μl sterile pipette tips. Then, the cells were washed with PBS, and fresh medium containing 10% FBS was added. After incubation for 24 hours at 37 °C, the cells were fixed and photographed. (F) The transwell migration assay showed the different migratory responses of the two stable cell lines. The cell lines were added to the upper chamber and 500 μl of appropriate medium supplemented with 10% FBS was added to the lower chamber. After incubation for 24 hours at 37 °C, the chambers were stained and photographed. Three random fields were analyzed for each chamber.
