The actin cortical meshwork and stress fiber were disrupted upon KAI1/CD82 expression.

<p>(<b><i>A</i></b><i>)</i> F-actin distribution. After being spread on FN (50 µg/ml)-, LN1 (50 µg/ml)-, or LN5 (2 µg/ml)-coated coverslips at 37°C, 5% CO₂ for 6 h, Du145 transfectant cells were fixed, permeabilized, and then stained with TRITC-conjugated α-phalloidin. The fluorescent images were captured under an Axiophot fluorescent microscope equipped with an Optronics digital camera at magnification 63X. (<b><i>B</i></b><i>)</i> Live imaging of actin polymerization in Du145-Mock and -KAI1/CD82 cells. The cells that were transiently transfected with pEGFP-actin construct were spread on FN-coated coverslips in complete DMEM and photographed using time-lapse confocal videomicroscopy. Arrowheads and arrows indicate actin polymerization during the development of peripheral meshwork and stress fiber, respectively. <i>(</i><b><i>C</i></b><i>)</i> Less F-actin in Du145-KAI1/CD82 cells. <i>Left panel</i>, Du145-Mock and -KAI1/CD82 cells spread in serum-free DMEM were detached, fixed, permeabilized, incubated with Alexa 488-conjugated phalloidin or mouse IgG2b, and then analyzed with flow cytometry. <i>Right top panel</i>, quantitation of the mean fluorescence intensity (MFI) of F-actin. Data are expressed as the mean MFI of three independent experiments (<i>P</i><0.05 between Mock and KAI1/CD82 cells). <i>Right bottom panel</i>, the cell lysates from the experiment were analyzed by Western blot for total cellular actin proteins. Cofilin blot was used as the protein loading control. <i>(</i><b><i>D</i></b><i>)</i> The presence of KAI1/CD82 proteins is inversely correlated with actin cytoskeletal assembly. HT29 colon cancer cells and prostate epithelial cells were fixed, permeabilized, and incubated with Alexa 488-conjugated phalloidin and Alexa 594-conjugated KAI1/CD82 mAb. The images were acquired with confocal microscopy. For HT29 cells, the photographs were taken from the basal sections of the cells. Shown in insets is the magnified staining from the frame-selected areas. White dashed lines outline the cell boundary. Scale bars: 10 µM. <i>(</i><b><i>E</i></b><i>)</i> KAI1/CD82 silencing leads to stress fiber formation. HT29 cells were transiently transfected with KAI1/CD82-specific siRNA duplexes, seeded sparsely, and stained with Alexa 488-conjugated phalloidin and Alexa 594-conjugated KAI1/CD82 mAb as described above. Scale bar: 10 µM. White dotted lines outline the cell periphery. Arrows indicate the cells exhibit robust stress fiber formation but no KAI1/CD82 staining.