Trict conjugated phalloidin

To visualize invadopodia matrix degradation, 5x10⁴ cells were embedded in 200 μl of Matrigel containing 25 μg/ml DQ-type I collagen (Life Technologies, Invitrogen) and mixed gently before plating in 8 wells Nunc^TM Lab-Tek^TM chambered coverglasses (Thermo Scientific) and incubating at 37°C for 2 hours. Then, medium supplemented with 10% FetalClone III and 2 ng/ml TGF-β was added on top of the Matrigel. After 72 hours, cells were fixed with 4% paraformaldehyde (PFA), permeabilized with 0.5% saponin, blocked with 5% bovine serum albumin (BSA), and F-actin was stained with TRICT-conjugated phalloidin (Sigma-Aldrich). Invadopodia were identified as actin-rich protrusions that colocalized with matrix degradations (enhanced DQ-type I collagen signal). 3D reconstructions were generated from Z-stacks using Volocity Software (Perkin Elmer, Waltham, MA).

1 × 10⁴ cells were seeded on glass coverslips in 12-well plates. After seeding, cells were serum starved for 24 hr. Cells were treated with 10μM lysophosphatidic acid for 30 minutes and then fixed with 4% paraformaldehyde in PBS and permeabilized with 0.2% Triton-X-100. F-actin was visualized by staining with TRICT conjugated phalloidin (Sigma Aldrich). Focal adhesions were stained with anti-paxillin antibody (Millipore). Nuclei were counter-stained with DAPI (Calbiochem). Immunofluorescence images were captured under Leica Q550CW fluorescence microscope (Leica) at 100× magnification. For scanning electron microscopy, cells were fixed with 2.5% glutaraldehyde and 1% osmium tetroxide. Fixed cells were then subjected to stepwise ethanol dehydration and critical point drying. Images were scanned and captured under 2,500× magnification using a Hitachi S-4800 FEG Scanning Electron Microscope (Hitachi).

Adult female flies were dissected in PBS. All the digestive tract was removed and fixed in PBS and 4% electron microscopy grade paraformaldehyde (Polysciences, USA) for 35 minutes. Samples were rinsed 3 times with PBS, 4% BSA, 0.1% Triton X-100 (PBT-BSA), incubated with the primary antibody overnight at 4°C and with the secondary antibody for 2 hours at room temperature. Finally, the samples were rinsed 3 times with PBT-BSA and mounted in DAPI-containing media (Vectashield, USA). All the steps were performed without mechanical agitation. Primary antibodies mouse α-Pros (1:100), α-Dl (1:10), α-Arm (1:10), α-Dlg (1:250) and α-Sn (1:50) were obtained from the Developmental Studies Hybridoma Bank (DSHB), rabbit α-PH 3 (1:100) from Cell Signalling (USA), rabbit α-Pdm1 (1:1000) was a gift of Dr.Yang Xiaohang (Institute of Molecular and Cell Biology, Singapore), rabbit α-laminin (1:1000) was from Sigma (USA) and goat α-GFP (1:500) was form Abcam (UK). Secondary antibodies were from Invitrogen (USA). TRICT-conjugated Phalloidin (Sigma, USA) was used at 5 µg/ml. Images were obtained on a Leica SPE or Leica SP5 confocal microscopy and processed in Photoshop CS5 (Adobe, USA).