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. Author manuscript; available in PMC: 2016 Jan 13.
Published in final edited form as: Cytoskeleton (Hoboken). 2014 Jun 26;71(6):380–394. doi: 10.1002/cm.21181

Figure 1. PMA treated Beas-2b cells form ventral actin waves.

Figure 1

(A) Untransfected Beas-2b cells were treated with vehicle (no PMA) or 200 nM PMA for 30 min prior to fixation and staining with rhodamine phalloidin. (B) Beas-2b cells were transfected with plasmids encoding untagged Arf6 Q67L (top) or Arf1 Q71L-GFP (bottom) and treated with 200 nM PMA for 30 min prior to fixation and labeling with antibodies to Arf6 for Arf6 detection and actin. Bars, 10 μm. (C) The fraction of transfected cells with 1 or more ventral waves was quantified and is expressed as the average percentage obtained from three independent experiments. Error bars represent standard deviation from the means. GFP was used as a control for transfection. One-way ANOVA test of the PMA-treated Control vs. Arf6Q67L- and Arf1Q71L-transfected cells were significant (p<0.05). (D) Beas-2b cells transfected with Mem-GFP and RFP-LifeAct were imaged as described in Materials and Methods. PMA was added after 2 min, designated time 0, and frames were captured every 30 seconds thereafter. Selected stills from the movie at −2, 6,12 and 18 min are shown (see Movie 1 in Supplementary materials). No membrane folds are associated with actin structures. Bar, 10μM. Boxed regions are shown at higher magnification below each time point. Bar, 5μM. (E) Beas-2b cells were transfected with plasmids encoding untagged Arf6 Q67L (in background), Mem-GFP to mark vacuolar membranes in transfected cells, and LifeAct-RFP (to visualize actin). A cell was imaged for 20 min; at 5 min, 200 nM PMA was added (arrow) to induce ventral wave formation (Movie 2 in Supplementary materials). (F) Beas-2b cells were transfected with plasmids encoding Arf1-Q71L-RFP and GFP-Actin. A cell was imaged for 15 minutes; at 5 min 200 nM PMA was added (arrow) to induce ventral wave formation (Movie 3 in Supplementary materials). Shown for each movie is an image of the entire cell taken at the end of the movie with a square around the region shown in the movie. A series of frames from the movie with time indicated in seconds from the beginning of the movie is also shown. Black arrows indicate when PMA was added. Bars,10 μm.