The following reagents were used: pig fibronectin (purified in our lab from pig plasma), fMLP (F3506; Sigma), cytochalasin D (250255; Calbiochem, San Diego, CA), blebbistatin (203389; Calbiochem), latrunculin B (428020; Calbiochem), jasplakinolide (420127; Calbiochem), Y27632 (688001; Calbiochem), and calcium dye, Fluo-4-AM (F14201; Invitrogen).
To test existing methods of arresting actin dynamics, the published concentrations of each drug were used (Kueh et al., 2008 (link); Renkawitz et al., 2009 (link); Wilson et al., 2010 (link)). For cytochalasin D experiments, cells were preincubated with 100 nM fMLP for a minimum of 2 min, and then a solution containing cytochalasin D and fMLP was added for a final concentration of 10 μM cytochalasin D and 100 nM fMLP. In experiments containing blebbistatin, cells were preincubated in 100 nM fMLP and 50 μM blebbistatin for 10 min. Cells were then treated with either solutions of latrunculin B or jasplakinolide to achieve final concentrations of 100 nM fMLP, 50 μM blebbistatin, and 500 nM latrunculin B, or 100 nM fMLP, 50 μM blebbistatin, and 1 μM jasplakinolide, respectively
For JLY treatment, HL-60 cells were preincubated in 100 nM fMLP and 10 μM Y27632 for 10 min. Next jasplakinolide and latrunculin B were added for a final concentration of 100 nM fMLP, 10 μM Y27632, 8 μM jasplakinolide, and 5 μM latrunculin B. In HT1080 and NIH 3T3 cells, the concentration of Y27632 in the cocktail was 20 μM Y27632. HT1080 cells were preincubated in 100 nM fMLP and 20 μM Y27632 for 10 min, and NIH 3T3 cells were preincubated in 20 μM Y27632 for 10 min before the addition of jasplakinolide and latrunculin B.
To test existing methods of arresting actin dynamics, the published concentrations of each drug were used (Kueh et al., 2008 (link); Renkawitz et al., 2009 (link); Wilson et al., 2010 (link)). For cytochalasin D experiments, cells were preincubated with 100 nM fMLP for a minimum of 2 min, and then a solution containing cytochalasin D and fMLP was added for a final concentration of 10 μM cytochalasin D and 100 nM fMLP. In experiments containing blebbistatin, cells were preincubated in 100 nM fMLP and 50 μM blebbistatin for 10 min. Cells were then treated with either solutions of latrunculin B or jasplakinolide to achieve final concentrations of 100 nM fMLP, 50 μM blebbistatin, and 500 nM latrunculin B, or 100 nM fMLP, 50 μM blebbistatin, and 1 μM jasplakinolide, respectively
For JLY treatment, HL-60 cells were preincubated in 100 nM fMLP and 10 μM Y27632 for 10 min. Next jasplakinolide and latrunculin B were added for a final concentration of 100 nM fMLP, 10 μM Y27632, 8 μM jasplakinolide, and 5 μM latrunculin B. In HT1080 and NIH 3T3 cells, the concentration of Y27632 in the cocktail was 20 μM Y27632. HT1080 cells were preincubated in 100 nM fMLP and 20 μM Y27632 for 10 min, and NIH 3T3 cells were preincubated in 20 μM Y27632 for 10 min before the addition of jasplakinolide and latrunculin B.
