Figure 4
Figure 4. CD151 silencing deregulates RhoA and Rac1 signaling. (A) Loss of CD151 expression results in increased stress fiber formation. After spreading overnight, HMEC transductants were stained with AlexaFluor-488-conjugated phalloidin. F-actin staining was visualized with a Zeiss LSM510 confocal fluorescence microscope under a 100×/1.4 NA oil objective. Bar represents 10 μm. (B) HMEC transductants were seeded in diluted Matrigel-coated dishes and cultured overnight. Cell lysates were incubated with glutathione-S-transferase-Rhotekin Rho-binding domain beads to pull down GTP-bound RhoA. RhoA levels in pull-down precipitates, and whole-cell lysates were determined by SDS-PAGE and immunoblotting using anti–human RhoA mAb. RhoAGTP/total RhoA ratio (right panel) was calculated from the density of 4 blots and normalized to the MOCK group. *P < .01. (C) Cellular ROCK enzymatic activities were measured using a commercially available immunoassay kit. Data from 4 independent experiments were normalized to the MOCK group. **P < .05. (D) HMEC cells were seeded as in the RhoA pulldown assay and lysed in RIPA buffer for 20 minutes at 4°C. After centrifugation, cell lysates were obtained and processed for SDS-PAGE and immunoblotting to detect total MLC and 2P-MLC. (E) GTP-bound Rac1 was precipitated from HMEC cell lysates using glutathione-S-transferase-PAK-1 Rac1-binding domain beads. Levels of Rac1GTP and total cellular Rac1 were determined by immunoblotting using Rac1 mAb (left panel). Rac1GTP/total Rac1 ratio was then calculated, normalized, and compared between MOCK and CD151 KD cells (right panel, n = 4). *P < .01. (F) RhoA signaling inhibitors rescue the defects in cell-cell adhesion and angiogenesis resulted from the loss of CD151. For the in vitro capillary-like network formation assay, various inhibitors (C3 transferase, 2.5 μg/mL; Y27632, 10μM; blebbistatin, 5μM; and ML-7, 5μM) were added at 4 hours after HMEC transductants (top) or MLECs (bottom) were plated on Matrigel. The capillary-like structures were photographed 14 hours later. Bar represents 250 μm.

CD151 silencing deregulates RhoA and Rac1 signaling. (A) Loss of CD151 expression results in increased stress fiber formation. After spreading overnight, HMEC transductants were stained with AlexaFluor-488-conjugated phalloidin. F-actin staining was visualized with a Zeiss LSM510 confocal fluorescence microscope under a 100×/1.4 NA oil objective. Bar represents 10 μm. (B) HMEC transductants were seeded in diluted Matrigel-coated dishes and cultured overnight. Cell lysates were incubated with glutathione-S-transferase-Rhotekin Rho-binding domain beads to pull down GTP-bound RhoA. RhoA levels in pull-down precipitates, and whole-cell lysates were determined by SDS-PAGE and immunoblotting using anti–human RhoA mAb. RhoAGTP/total RhoA ratio (right panel) was calculated from the density of 4 blots and normalized to the MOCK group. *P < .01. (C) Cellular ROCK enzymatic activities were measured using a commercially available immunoassay kit. Data from 4 independent experiments were normalized to the MOCK group. **P < .05. (D) HMEC cells were seeded as in the RhoA pulldown assay and lysed in RIPA buffer for 20 minutes at 4°C. After centrifugation, cell lysates were obtained and processed for SDS-PAGE and immunoblotting to detect total MLC and 2P-MLC. (E) GTP-bound Rac1 was precipitated from HMEC cell lysates using glutathione-S-transferase-PAK-1 Rac1-binding domain beads. Levels of Rac1GTP and total cellular Rac1 were determined by immunoblotting using Rac1 mAb (left panel). Rac1GTP/total Rac1 ratio was then calculated, normalized, and compared between MOCK and CD151 KD cells (right panel, n = 4). *P < .01. (F) RhoA signaling inhibitors rescue the defects in cell-cell adhesion and angiogenesis resulted from the loss of CD151. For the in vitro capillary-like network formation assay, various inhibitors (C3 transferase, 2.5 μg/mL; Y27632, 10μM; blebbistatin, 5μM; and ML-7, 5μM) were added at 4 hours after HMEC transductants (top) or MLECs (bottom) were plated on Matrigel. The capillary-like structures were photographed 14 hours later. Bar represents 250 μm.

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