Plan apochromatic 100 silicone

After 96 h of induction, cells were fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) and permeabilized using 0.1% Triton-X. Nonspecific interactions were blocked using 10% normal goat serum in PBS. The cells were then incubated overnight with the primary antibody (anti-paxillin 1:50 and anti-ERM 1:100), washed in PBS, and then followed by incubation with the appropriate Alexa Fluor 647 secondary antibody (1:200) in 10% normal goat serum for 1 h at room temperature. In experiments where phalloidin or membrane stain was used, it was added after the secondary at 1:1000 in PBS and incubated for 30 min at room temperature. Cells were washed 3× in PBS and then imaged using the same parameters as previously described using a Plan Apochromatic 100× silicone oil immersion objective (Nikon) but at room temperature with no CO₂.

Cells were treated with the actin inhibitor cytochalasin-D (5 µM; Tocris), the ezrin inhibitor NSC668394 (50 µM; Calbiochem), Y27632 (25 µM; Hello Bio), calyculin A (10 nM; Cayman Chemical), Ouabain (30 nM; Tocris), and s-nitro-Blebbistatin 25 µM; Cayman Chemical). For cytochalasin-D and calyculin A treatment, an 18-slice Z-stack (140 nm step size) was acquired every minute for 5 min using a Plan Apochromatic 100× silicone oil immersion objective (Nikon; NA 1.35) as described above. After 5 min, a solution of cytochalasin-D dissolved in imaging media was injected into the well and the cells were imaged for another 15 min posttreatment. For NSC668394, Y27632, Ouabain, and S-nitro- Blebbistatin, cells were treated immediately before imaging and an 18-slice Z-stack (192.5 nm step size) was acquired every hour for 17 h using a Plan Apochromatic 40× air objective (Nikon; NA 0.95) and a 2× zoom lens (80× total magnification). Each drug treatment experiment was repeated in three separate wells, and multiple fields of view were collected per well.