Figure 3
Figure 3. RAC2 is associated with cytoskeletal reorganization and invasion by SD1 cells. (A) Organized filamentous actin is present in SD1 cells compared with REH and SupB15. Cells were stained with Alexa Fluor 555 phalloidin (red); DAPI stains the nuclei. Trans refers to bright-field images. Original magnification is ×100. Scale bar indicates 5 μm. (B) Whereas all cell lines expressed RAC1, SD1 alone expressed detectable active RAC2 (left panel). Invasion by SD1 cells was significantly (P < .0001) inhibited by preincubation with a RAC inhibitor (RACi, 100μM; right panel). (C) SD1RAC2kd cells expressed RAC1 but not RAC2 (left panel) and exhibited significantly decreased in vitro invasion compared with SD1 (P = .009; right panel). Dashed lines denote spliced noncontiguous lines within gels. (D) Conceptual model of proteins identified in Table 1 suggesting that adhesion is mediated by ICAM1 and LFA-1 as part of a RAC2-initiated invasion process. (E) All 3 cell line membranes were enriched in lipid rafts, most prominently in SD1. In addition, in SD1, lipid raft–enriched peripheral vesicles are seen (arrow). Lipid rafts were labeled with the fluorophore-tagged cholera toxin B subunit (green); DAPI stains the nucleus. Original magnification is ×100. Scale bar indicates 10μm. For panels A and E, data were viewed and captured using a low light imaging system based around a Zeiss Axiovert 200M microscope using a Zeiss α Plan-Fluor 100×/1.45 numerical aperture (NA) objective lens and an Andor iXon3 EMCCD 888 camera. Using Applied Scientific Imaging MS2000, stage focus and volume imaging were attained. The system illumination was controlled via Sutter λ 10-3 and associated filter wheels and light sources were a Sutter Lambda-LS 300W Xenon UV light source and a Thorlabs white light LED for transmitted light. UV filters used were the Chroma ET-Sedat filter set. The equipment was enclosed in an environmental chamber and for fixed cell imaging, all work was carried out at 25°C. The equipment was controlled via Metamorph software (Molecular Devices).

RAC2 is associated with cytoskeletal reorganization and invasion by SD1 cells. (A) Organized filamentous actin is present in SD1 cells compared with REH and SupB15. Cells were stained with Alexa Fluor 555 phalloidin (red); DAPI stains the nuclei. Trans refers to bright-field images. Original magnification is ×100. Scale bar indicates 5 μm. (B) Whereas all cell lines expressed RAC1, SD1 alone expressed detectable active RAC2 (left panel). Invasion by SD1 cells was significantly (P < .0001) inhibited by preincubation with a RAC inhibitor (RACi, 100μM; right panel). (C) SD1RAC2kd cells expressed RAC1 but not RAC2 (left panel) and exhibited significantly decreased in vitro invasion compared with SD1 (P = .009; right panel). Dashed lines denote spliced noncontiguous lines within gels. (D) Conceptual model of proteins identified in Table 1 suggesting that adhesion is mediated by ICAM1 and LFA-1 as part of a RAC2-initiated invasion process. (E) All 3 cell line membranes were enriched in lipid rafts, most prominently in SD1. In addition, in SD1, lipid raft–enriched peripheral vesicles are seen (arrow). Lipid rafts were labeled with the fluorophore-tagged cholera toxin B subunit (green); DAPI stains the nucleus. Original magnification is ×100. Scale bar indicates 10μm. For panels A and E, data were viewed and captured using a low light imaging system based around a Zeiss Axiovert 200M microscope using a Zeiss α Plan-Fluor 100×/1.45 numerical aperture (NA) objective lens and an Andor iXon3 EMCCD 888 camera. Using Applied Scientific Imaging MS2000, stage focus and volume imaging were attained. The system illumination was controlled via Sutter λ 10-3 and associated filter wheels and light sources were a Sutter Lambda-LS 300W Xenon UV light source and a Thorlabs white light LED for transmitted light. UV filters used were the Chroma ET-Sedat filter set. The equipment was enclosed in an environmental chamber and for fixed cell imaging, all work was carried out at 25°C. The equipment was controlled via Metamorph software (Molecular Devices).

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