1.3na oil objective

To visualize focal adhesions, A549 and H460 cells grown on fibronectin-coated coverslips were treated with vehicle (DMSO) or EL (100 μM) for 24 h. After treatment, the cells were washed with PBS, fixed with 4% formaldehyde solution for 15 min at room temperature, and permeabilized with 0.1% Triton-X 100 for 10 min. Next, the cells were washed with 1x TBS-tween (0.1%),blocked with 10% NGS for 1 h, washed with 1x TBS-tween (0.1%), and incubated with anti-vinculin primary antibody for 3 h. The cells were washed again with 1x TBS-tween (0.1%) and incubated with Alexa Fluor 633 anti-mouse secondary antibody for 1 h. These cells were then incubated with DAPI for 3–4 min to label nuclei. The coverslips were then mounted onto slides with the help of aqua-poly-mount mounting medium and examined using Zeiss Axio Observer Z1 inverted microscope with LSM700 laser scanning unit and 40x 1.3NA oil objective (Zeiss, Thornwood, NY). The number and size of focal adhesions per cell were examined analyzed using the ImagePro Premier software (Media Cybernetics, Silver Spring, MD, USA) in 10 individual cells for each treatment for three independent experiments.

Images were acquired using Zeiss LSM900 confocal microscope, 40X/1.3 NA oil objective and 0.8x zoom. All images were acquired with the “confocal” setting for optimal pixel sampling of the regions of interests. All the images were acquired at 16 bits, without any averaging across pixels. Optical slices were designated at a constant interval of 0.45 μm, from the bottom of the cell to the top. Depending on the spread of cells on coverslips, the number of optical sections acquired ranged between 15 and 30. 3-D rendering of confocal images were done using Imaris (9.9.1). Snapshots of 3-D view are represented in Figs. 4E and 5G-H.