Data were analyzed using TRI2 software. All histogram data were plotted as mean FRET efficiency from 10 cells per sample. Lifetime images of exemplary cells are presented using a pseudocolor scale, with blue depicting normal GFP lifetime and red depicting reduced GFP lifetime. Each experiment was repeated at least three following website times. Live cell imaging SW480 cells were transfected with GFP fascin 1, and 24 hours later, were plated onto glass bottomed imaging chambers coated with 15 nmoll laminin for 6 hours before treatment with DMSO or 10 umoll Y27632 for 30 minutes. Live confocal microscopy imaging was performed on a microscope equipped with a 40 oil objective lens at 37 C. Images were captured and exported with NIS Elements software.
Movies were acquired as one image per 2 sec onds over 60 to 150 frames, and are presented at play back rates of 15 framessecond. Morphometric analyses of filopodia formation in SW480 cells were performed by measuring the number and length of GFP tagged or RFP tagged fascin positive filopodia, and by line scan analysis of fluorescence intensity along the length of single, ran Inhibitors,Modulators,Libraries domly selected filopodia. To analyze the dynamics of an individual filopodium, kymographs of 210 um areas encompassing one filopodium were selected, and the montage was performed in the X axis. The mRFP fascin 1 signal was thresholded, the front of the Inhibitors,Modulators,Libraries filopodium out lined, and the resulting coordinates used to calculate sev eral dynamic values including maximum filopodia displacement. All image analyses were performed with ImageJ software.
Each experimental condition was tested in at least three independent experiments. Immunoblotting and fascin 1 pull downs Adherent cells for immunoblotting were extracted on ice in 1% Triton X 100 in TBS containing protease inhi bitor Inhibitors,Modulators,Libraries cocktail for 12 minutes. SDS PAGE and immunoblotting with selected antibodies and enhanced chemiluminescence Inhibitors,Modulators,Libraries detection was carried out as described previously. Recombinant 6His tagged fascin 1 was expressed in BL21 Inhibitors,Modulators,Libraries E. coli by induction with 1 mmoll isopropyl b D 1 hiogalactopyranoside for 5 hours at 25 C. Bacterial pellets were resuspended in buf fer, then sonicated and clarified. Lysates were passed over nickel nitrilotriacetic acid beads to allow fascin 1 binding, beads were washed, and fascin 1 was eluted, and dialyzed against buffer. For pull down experiments, the wild type or mutant fascin 1 proteins were immobilized Alisertib on Ni NTA beads in disposable polystyrene chromatogra phy columns. SW480 cell lysates were extracted, then passed through the columns, washed extensively, and the bound proteins were eluted. The eluted proteins were mixed with SDS sample buffer and subjected to SDS PAGE and immunoblotting as described previously.