Figure 3
Figure 3. DMP1 induces CD44-dependent VE-cadherin expression and mediates inhibition of growth in sparse HUVECs. (A) Immunofluorescence microscopy of sparse HUVECs treated with DMP1 (50 nmol/L) for 3 and 24 hours. Nuclei appear blue after TO-PRO-3 staining. Representative confocal fields of one experiment (n = 3) are shown (magnification, 40×). (B) Western blot analysis with antibodies to VE-cadherin and αV-integrin using membrane lysates from DMP1-treated HUVECs. (C) Western blot analysis with antibodies to p27Kip1 and VE-cadherin using total lysates from HUVECs transfected for 48 hours with VE-cadherin or nontargeting siRNAs and treated with DMP1 (50 nmol/L) for the last 24 hours of transfection. (D) Western blot analysis with an antibody to VE-cadherin using total lysates from HUVECs incubated for 1 hour in the presence of 10 μg/mL of anti-CD44 blocking antibody or IgG before DMP1 treatment (50 nmol/L) for 24 hours. (E) Western blot analysis with antibodies to VE-cadherin and p27Kip1 using total lysates from sparse and confluent DPM1-treated HUVECs, as described in “Methods.” All Western blotting results were evaluated by densitometric scanning. The relative protein level values are shown in italics below the lanes. Western blots were performed 3 times with similar results. Equal protein loading was assessed by anti–β-actin immunoblotting. (F) Immunofluorescence of sparse and confluent DMP1-treated HUVECs incubated with BrdU for 20 hours to evaluate the S-phase population, as described in “Methods.” VE-cadherin, BrdU, and phalloidin are shown in green, red, and gray, respectively. As expected, control sparse cells presented with S-phase–positive and VE-cadherin–negative staining compared with control confluent cells. DMP1-treated sparse cells showed strong positive VE-cadherin staining and less BrdU incorporation than control cells, similar to that of control or DMP1-treated confluent cells. Confluent cells did not show any modulation of VE-cadherin staining intensity or BrdU incorporation after DMP1 treatment. Representative confocal fields of one experiment (n = 3) are shown (magnification, 40×).

DMP1 induces CD44-dependent VE-cadherin expression and mediates inhibition of growth in sparse HUVECs. (A) Immunofluorescence microscopy of sparse HUVECs treated with DMP1 (50 nmol/L) for 3 and 24 hours. Nuclei appear blue after TO-PRO-3 staining. Representative confocal fields of one experiment (n = 3) are shown (magnification, 40×). (B) Western blot analysis with antibodies to VE-cadherin and αV-integrin using membrane lysates from DMP1-treated HUVECs. (C) Western blot analysis with antibodies to p27Kip1 and VE-cadherin using total lysates from HUVECs transfected for 48 hours with VE-cadherin or nontargeting siRNAs and treated with DMP1 (50 nmol/L) for the last 24 hours of transfection. (D) Western blot analysis with an antibody to VE-cadherin using total lysates from HUVECs incubated for 1 hour in the presence of 10 μg/mL of anti-CD44 blocking antibody or IgG before DMP1 treatment (50 nmol/L) for 24 hours. (E) Western blot analysis with antibodies to VE-cadherin and p27Kip1 using total lysates from sparse and confluent DPM1-treated HUVECs, as described in “Methods.” All Western blotting results were evaluated by densitometric scanning. The relative protein level values are shown in italics below the lanes. Western blots were performed 3 times with similar results. Equal protein loading was assessed by anti–β-actin immunoblotting. (F) Immunofluorescence of sparse and confluent DMP1-treated HUVECs incubated with BrdU for 20 hours to evaluate the S-phase population, as described in “Methods.” VE-cadherin, BrdU, and phalloidin are shown in green, red, and gray, respectively. As expected, control sparse cells presented with S-phase–positive and VE-cadherin–negative staining compared with control confluent cells. DMP1-treated sparse cells showed strong positive VE-cadherin staining and less BrdU incorporation than control cells, similar to that of control or DMP1-treated confluent cells. Confluent cells did not show any modulation of VE-cadherin staining intensity or BrdU incorporation after DMP1 treatment. Representative confocal fields of one experiment (n = 3) are shown (magnification, 40×).

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