Journal: BMC Cancer

Article Title: KAI1 suppresses HIF-1α and VEGF expression by blocking CDCP1-enhanced Src activation in prostate cancer

doi: 10.1186/1471-2407-12-81

Figure Lengend Snippet: KAI1-mediated negative regulation of CDCP1 inhibits Src . ( A ) Vector-transfected cells (PC3-GFP #8) and KAI1-expressing PC3 clones (PC3-KAI1 #6) were transiently transfected with siRNA against integrin β1. Forty-eight hours after transfection, the levels of phospho-Src Y416 , phospho-FAK Y397 , phospho-p130Cas Y410 , and integrin β1 were measured by immunoblotting. Densitometric analysis of phospho-Src Y416 was shown after normalization to beta-actin. ( B ) PC3 cells were transfected with KAI1. Twenty-four hours after transfection, phospho-Src Y416 and CDCP1 were detected by immunoblotting. Densitometric analysis of CDCP1 was shown after normalization to beta-actin. ( C ) Cell lysates from PC3 vector control (PC3-GFP #8) and KAI1 transfectants (PC3-KAI1 #5 and PC3-KAI1 #6) were analyzed for CDCP1, phospho-Src Y416 , PKCδ, integrin β1, and GFP-KAI1 protein levels. Densitometric analysis of CDCP1 was shown after normalization to beta-actin. ( D ) CDCP1 proteins were detected by immunofluorescence using anti-CDCP1 antibody and visualized using rhodamine-labeled secondary antibody. ( E ) PC3 cells stably transfected with KAI1 (PC3-KAI1 #6) and vector control (PC3-GFP #8) cells were treated with siRNA against KAI1. After 48 hours, cell lysates were analyzed by immunoblotting to detect CDCP1 and GFP-KAI1. ( F ) After siRNA-mediated knockdown of CDCP1, phospho-Src Y416 levels were monitored by immunoblotting.

Article Snippet: The slides were then washed in Tris-NaCl buffer and incubated at room temperature for 60 minutes with anti-CDCP1 antibody (R&D systems, Minneapolis, MN, USA) at a 1:50 dilution, anti-HIF-1 antibody (Thermo Fisher Scientific, MA, USA) at a 1:50 dilution, anti-VHL antibody (Thermo Fisher Scientific, MA, USA) at a 1:50 dilution.

Techniques: Plasmid Preparation, Transfection, Expressing, Clone Assay, Western Blot, Control, Immunofluorescence, Labeling, Stable Transfection, Knockdown

Journal: BMC Cancer

Article Title: KAI1 suppresses HIF-1α and VEGF expression by blocking CDCP1-enhanced Src activation in prostate cancer

doi: 10.1186/1471-2407-12-81

Figure Lengend Snippet: KAI1 increases VHL protein levels . ( A ) PC3 cells stably transfected with KAI1 (PC3-KAI1 #6) or vector control (PC3-GFP #8) cells were treated with MG132. After 12 hours of treatment, cell lysates were prepared and the levels of CDCP1 were measured by immunoblotting. ( B ) Cell lysates were prepared from PC3 cells transiently transfected with KAI1 and from stable transfectants. VHL expression was analyzed by immunoprecipitation. Densitometric analysis of VHL was shown after normalization to beta-actin. ( C ) PC3 cells stably transfected with KAI1 (PC3-KAI1 #5 and PC3-KAI1 #6) or vector control (PC3-GFP #8) were transiently transfected with HA-tagged VHL. Twenty-four hours after transfection, cells were harvested, and the levels of CDCP1 and VHL protein were analyzed by immunoblotting. Densitometric analysis of HA-VHL was shown after normalization to beta-actin. ( D ) PC3 cells were transfected with HA-tagged VHL, and then treated with siRNA against VHL. The protein levels of CDCP1 and HA-VHL were assessed by immunoblotting.

Article Snippet: The slides were then washed in Tris-NaCl buffer and incubated at room temperature for 60 minutes with anti-CDCP1 antibody (R&D systems, Minneapolis, MN, USA) at a 1:50 dilution, anti-HIF-1 antibody (Thermo Fisher Scientific, MA, USA) at a 1:50 dilution, anti-VHL antibody (Thermo Fisher Scientific, MA, USA) at a 1:50 dilution.

Techniques: Stable Transfection, Transfection, Plasmid Preparation, Control, Western Blot, Expressing, Immunoprecipitation

Journal: BMC Cancer

Article Title: KAI1 suppresses HIF-1α and VEGF expression by blocking CDCP1-enhanced Src activation in prostate cancer

doi: 10.1186/1471-2407-12-81

Figure Lengend Snippet: KAI1 blocks HIF-1α induction . ( A ) After transfection of KAI1, cells were treated with 100 μM CoCl 2 for 24 hours. PC3 vector control (PC3-GFP #8) and KAI1 transfectants (PC3-KAI1 #5 and PC3-KAI1 #6) were also treated with 100 μM of CoCl 2 for 24 hours. Cells were harvested and immunoblotted for detection of HIF-1α and GFP-KAI1. Densitometric analysis of HIF-1α was shown after normalization to beta-actin. ( B ) PC3 vector control (PC3-GFP #8) and KAI1 transfectants (PC3-KAI1 #5 and PC3-KAI1 #6) were exposed to hypoxia for 24 hours. The levels of HIF-1α, CDCP1, and GFP-KAI1 proteins were assessed by immunoblotting. Densitometric analysis of HIF-1α was shown after normalization to beta-actin. HIF-1α and CDCP1 mRNA levels were measured by RT-PCR. ( C ) PC3 cells were cotransfected with KAI1 and HIF-1α, then treated with the proteasome inhibitor, MG132. HIF-1α levels were assessed by immunoblotting. ( D ) Stable KAI1-expressing (PC3-KAI1 #6) and vector control (PC3-GFP #8) PC3 cell clones were treated with siRNA against CDCP1 and VHL. Forty-eight hours after transfection, HIF-1α, CDCP1, and VHL protein levels were measured by immunoblotting.

Article Snippet: The slides were then washed in Tris-NaCl buffer and incubated at room temperature for 60 minutes with anti-CDCP1 antibody (R&D systems, Minneapolis, MN, USA) at a 1:50 dilution, anti-HIF-1 antibody (Thermo Fisher Scientific, MA, USA) at a 1:50 dilution, anti-VHL antibody (Thermo Fisher Scientific, MA, USA) at a 1:50 dilution.

Techniques: Transfection, Plasmid Preparation, Control, Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing, Clone Assay

Journal: BMC Cancer

Article Title: KAI1 suppresses HIF-1α and VEGF expression by blocking CDCP1-enhanced Src activation in prostate cancer

doi: 10.1186/1471-2407-12-81

Figure Lengend Snippet: KAI1 decreases VEGF expression . ( A ) PC3 cells were cotransfected with a VEGF promoter (2.2 kb)-luciferase reporter plasmid and HIF-1α and GFP-KAI1 expression plasmids. Forty-eight hours after transfection, cell lysates were analyzed for luciferase expression. ( B ) PC3 cells stably transfected with KAI1 (PC3-KAI1 #5 and PC3-KAI1 #6) or vector control (PC3-GFP #8) were cotransfected with a VEGF promoter-luciferase reporter plasmid and a HIF-1α expression vector. Forty-eight hours after transfection, cell lysates were analyzed for luciferase activity. ( C ) PC3 cells were cotransfected with a VEGF promoter-luciferase reporter plasmid and a GFP-KAI1 expression plasmid. Twenty-four hours after transfection, cells were treated with 100 μM CoCl 2 for 24 hours, and then luciferase activity was measured. ( D ) Total RNA from PC3 cells stably transfected with KAI1 (PC3-KAI1 #5 and PC3-KAI1 #6) or vector control (PC3-GFP #8) was isolated, and VEGF mRNA expression was analyzed by RT-PCR. ( E ) After treatment of si-CDCP1, PC3 cells were harvested and VEGF mRNA expression was analyzed by RT-PCR. ( F ). PC3 cells stably transfected with KAI1 (#6) or vector control (PC3-GFP #8) were exposed to CoCl 2 (100 μM) for 24 hours. Total RNA was extracted and mRNA levels were quantified by RT-PCR.

Article Snippet: The slides were then washed in Tris-NaCl buffer and incubated at room temperature for 60 minutes with anti-CDCP1 antibody (R&D systems, Minneapolis, MN, USA) at a 1:50 dilution, anti-HIF-1 antibody (Thermo Fisher Scientific, MA, USA) at a 1:50 dilution, anti-VHL antibody (Thermo Fisher Scientific, MA, USA) at a 1:50 dilution.

Techniques: Expressing, Luciferase, Plasmid Preparation, Transfection, Stable Transfection, Control, Activity Assay, Isolation, Reverse Transcription Polymerase Chain Reaction

Journal: BMC Cancer

Article Title: KAI1 suppresses HIF-1α and VEGF expression by blocking CDCP1-enhanced Src activation in prostate cancer

doi: 10.1186/1471-2407-12-81

Figure Lengend Snippet: KAI1 inhibits HIF-1α and CDCP1 expression and VEGF secretion in tumor xenografts . ( A ) PC3 cells stably transfected with KAI1 (PC3-KAI1 #5 andPC3-KAI1 #6) or vector control (PC3-GFP #8) were resuspended in PBS (2 × 10 6 cells/100 μl) and injected subcutaneously into the flanks of athymic nude mice. Animals were sacrificed 35 days after injection, at which time tumors were collected and photographed. Tumor volumes were measured using precision calipers ( B ) Photomicrographs showing expression of HIF-1α, CDCP1, and VHL protein in tumor sections by immunohistochemistry.

Article Snippet: The slides were then washed in Tris-NaCl buffer and incubated at room temperature for 60 minutes with anti-CDCP1 antibody (R&D systems, Minneapolis, MN, USA) at a 1:50 dilution, anti-HIF-1 antibody (Thermo Fisher Scientific, MA, USA) at a 1:50 dilution, anti-VHL antibody (Thermo Fisher Scientific, MA, USA) at a 1:50 dilution.

Techniques: Expressing, Stable Transfection, Transfection, Plasmid Preparation, Control, Injection, Immunohistochemistry