Journal: Cancer Research

Article Title: Down-regulation of Platelet-Derived Growth Factor-D Inhibits Cell Growth and Angiogenesis through Inactivation of Notch-1 and Nuclear Factor-κB Signaling

doi: 10.1158/0008-5472.can-07-2803

Figure Lengend Snippet: Figure 2. PDGF-D expression in pancreatic cancer cell lines. CS, control siRNA; PS, PDGF-D siRNA; CP, control plasmid; PP, PDGF-D cDNA plasmid. A and B, PDGF-D (top) and PDGFRh (bottom) mRNA levels were measured by real-time RT-PCR in seven pancreatic cancer cell lines and stable PDGF-D–transfected cell lines, respectively. C, PDGF-D protein level was measured by Western blotting in seven pancreatic cancer cell lines and PDGF-D–transfected cell lines. Columns, mean of three independent experiments; bars, SD. *, P < 0.05, relative to control.

Article Snippet: The PDGF-D cDNA plasmid was purchased from OriGene Technologies, Inc. Human pancreatic cancer cells were transfected with PDGF-D siRNA and cDNA, respectively, using Lipofectamine 2000 as described earlier (15).

Techniques: Expressing, Control, Plasmid Preparation, Quantitative RT-PCR, Transfection, Western Blot

Journal: Cancer Research

Article Title: Down-regulation of Platelet-Derived Growth Factor-D Inhibits Cell Growth and Angiogenesis through Inactivation of Notch-1 and Nuclear Factor-κB Signaling

doi: 10.1158/0008-5472.can-07-2803

Figure Lengend Snippet: Figure 3. Effects of altered PDGF-D expression on pancreatic cancer cell growth and apoptosis. A, inhibition of cancer cell growth by PDGF-D siRNA as measured by MTT assay. B, induction of cancer cell apoptotic death by PDGF-D siRNA as measured by ELISA. C, promotion of cancer cell growth by PDGF-D cDNA as measured by MTT assay. Points, mean of three separate experiments having six determinations per experiment for each experimental condition; bars, SD. *, P < 0.05; **, P < 0.01, relative to control.

Article Snippet: The PDGF-D cDNA plasmid was purchased from OriGene Technologies, Inc. Human pancreatic cancer cells were transfected with PDGF-D siRNA and cDNA, respectively, using Lipofectamine 2000 as described earlier (15).

Techniques: Expressing, Inhibition, MTT Assay, Enzyme-linked Immunosorbent Assay, Control

Journal: Cancer Research

Article Title: Down-regulation of Platelet-Derived Growth Factor-D Inhibits Cell Growth and Angiogenesis through Inactivation of Notch-1 and Nuclear Factor-κB Signaling

doi: 10.1158/0008-5472.can-07-2803

Figure Lengend Snippet: Figure 4. Effects of altered PDGF-D expression on Notch-1 expression in human pancreatic cancer cells. A, the expression of Notch-1 and its target genes was detected by Western blotting. The expression of selected proteins was inhibited by PDGF-D siRNA and increased by PDGF-D cDNA transfection, respectively. B, the Notch-1 mRNA level was increased by PDGF-D cDNA transfection as measured by real-time RT-PCR. C, the PDGF-D cDNA– transfected cells were subjected to immunofluorescent staining using anti-Notch-1 antibody. Higher level of Notch-1 protein in the nucleus was found in the PDGF-D–transfected cells. DAPI, 4¶,6-diamidino-2-phenylindole.

Article Snippet: The PDGF-D cDNA plasmid was purchased from OriGene Technologies, Inc. Human pancreatic cancer cells were transfected with PDGF-D siRNA and cDNA, respectively, using Lipofectamine 2000 as described earlier (15).

Techniques: Expressing, Western Blot, Transfection, Quantitative RT-PCR, Staining

Journal: Cancer Research

Article Title: Down-regulation of Platelet-Derived Growth Factor-D Inhibits Cell Growth and Angiogenesis through Inactivation of Notch-1 and Nuclear Factor-κB Signaling

doi: 10.1158/0008-5472.can-07-2803

Figure Lengend Snippet: Figure 5. A, nuclear proteins from siRNA- and cDNA-transfected cells were subjected to analysis for NF-nB DNA-binding activity as measured by EMSA. Left, down-regulation of PDGF-D inhibited NF-nB DNA-binding activity compared with control; middle, PDGF-D cDNA transfection caused activation of NF-nB DNA-binding activity in all three cell lines tested; right, NF-nB supershift analyses. EMSA experiments were done by additional 30-min incubations with polyclonal supershift antibodies against p65 before the addition of labeled probe. Lane 1, nonspecific antibody (anti-cyclin D1); lane 2, p65 antibody. B, left, Western blot analysis showed that PDGF-D cDNA transfection increased the expression of MMP-9 and VEGF; right, real-time RT-PCR showed that PDGF-D cDNA increased the expression of MMP-9 genes at mRNA level in pancreatic cancer cells. C, left, PDGF-D cDNA transfection increased the activity of MMP-9 in pancreatic cancer cells; right, PDGF-D cDNA transfection increased the secreted levels of VEGF in pancreatic cancer cells. *, P < 0.05; **, P < 0.01, relative to control.

Article Snippet: The PDGF-D cDNA plasmid was purchased from OriGene Technologies, Inc. Human pancreatic cancer cells were transfected with PDGF-D siRNA and cDNA, respectively, using Lipofectamine 2000 as described earlier (15).

Techniques: Transfection, Binding Assay, Activity Assay, Control, Activation Assay, Labeling, Western Blot, Expressing, Quantitative RT-PCR

Journal: Cancer Research

Article Title: Down-regulation of Platelet-Derived Growth Factor-D Inhibits Cell Growth and Angiogenesis through Inactivation of Notch-1 and Nuclear Factor-κB Signaling

doi: 10.1158/0008-5472.can-07-2803

Figure Lengend Snippet: Figure 6. PDGF-D cDNA transfection increased pancreatic cancer cell migration and invasion and induced the HUVEC tube formation. *, P < 0.05, relative to control. A, top, migration assay showing that PDGF-D cDNA transfection increased pancreatic cancer cell migration; bottom, value of fluorescence from the migrated cells. B, top, invasion assay showing that PDGF-D cDNA transfection resulted in high penetration of cells through the Matrigel-coated membrane compared with control cells; bottom, value of fluorescence of the invaded cells. The values indicated the comparative levels of invaded cells. C, top, conditioned media from PDGF-D cDNA–transfected BxPC-3 and MIA PaCa cells were able to significantly induce the tube formation of HUVECs in 6-h incubation compared with the conditioned medium from control cells; bottom, image analysis of tubule/capillary length was carried out using software image analysis program Scion Image. Quantification of cumulative tube length of endothelial cells.

Article Snippet: The PDGF-D cDNA plasmid was purchased from OriGene Technologies, Inc. Human pancreatic cancer cells were transfected with PDGF-D siRNA and cDNA, respectively, using Lipofectamine 2000 as described earlier (15).

Techniques: Transfection, Migration, Control, Fluorescence, Invasion Assay, Membrane, Incubation, Software

Journal: Cancer Research

Article Title: Down-regulation of Platelet-Derived Growth Factor-D Inhibits Cell Growth and Angiogenesis through Inactivation of Notch-1 and Nuclear Factor-κB Signaling

doi: 10.1158/0008-5472.can-07-2803

Figure Lengend Snippet: Figure 2. PDGF-D expression in pancreatic cancer cell lines. CS, control siRNA; PS, PDGF-D siRNA; CP, control plasmid; PP, PDGF-D cDNA plasmid. A and B, PDGF-D (top) and PDGFRh (bottom) mRNA levels were measured by real-time RT-PCR in seven pancreatic cancer cell lines and stable PDGF-D–transfected cell lines, respectively. C, PDGF-D protein level was measured by Western blotting in seven pancreatic cancer cell lines and PDGF-D–transfected cell lines. Columns, mean of three independent experiments; bars, SD. *, P < 0.05, relative to control.

Article Snippet: PDGF-D siRNA and control siRNA were obtained from Santa Cruz Biotechnology.

Techniques: Expressing, Control, Plasmid Preparation, Quantitative RT-PCR, Transfection, Western Blot

Journal: Cancer Research

Article Title: Down-regulation of Platelet-Derived Growth Factor-D Inhibits Cell Growth and Angiogenesis through Inactivation of Notch-1 and Nuclear Factor-κB Signaling

doi: 10.1158/0008-5472.can-07-2803

Figure Lengend Snippet: Figure 3. Effects of altered PDGF-D expression on pancreatic cancer cell growth and apoptosis. A, inhibition of cancer cell growth by PDGF-D siRNA as measured by MTT assay. B, induction of cancer cell apoptotic death by PDGF-D siRNA as measured by ELISA. C, promotion of cancer cell growth by PDGF-D cDNA as measured by MTT assay. Points, mean of three separate experiments having six determinations per experiment for each experimental condition; bars, SD. *, P < 0.05; **, P < 0.01, relative to control.

Article Snippet: PDGF-D siRNA and control siRNA were obtained from Santa Cruz Biotechnology.

Techniques: Expressing, Inhibition, MTT Assay, Enzyme-linked Immunosorbent Assay, Control

Journal: Cancer Research

Article Title: Down-regulation of Platelet-Derived Growth Factor-D Inhibits Cell Growth and Angiogenesis through Inactivation of Notch-1 and Nuclear Factor-κB Signaling

doi: 10.1158/0008-5472.can-07-2803

Figure Lengend Snippet: Figure 4. Effects of altered PDGF-D expression on Notch-1 expression in human pancreatic cancer cells. A, the expression of Notch-1 and its target genes was detected by Western blotting. The expression of selected proteins was inhibited by PDGF-D siRNA and increased by PDGF-D cDNA transfection, respectively. B, the Notch-1 mRNA level was increased by PDGF-D cDNA transfection as measured by real-time RT-PCR. C, the PDGF-D cDNA– transfected cells were subjected to immunofluorescent staining using anti-Notch-1 antibody. Higher level of Notch-1 protein in the nucleus was found in the PDGF-D–transfected cells. DAPI, 4¶,6-diamidino-2-phenylindole.

Article Snippet: PDGF-D siRNA and control siRNA were obtained from Santa Cruz Biotechnology.

Techniques: Expressing, Western Blot, Transfection, Quantitative RT-PCR, Staining

Journal: Cancer Research

Article Title: Down-regulation of Platelet-Derived Growth Factor-D Inhibits Cell Growth and Angiogenesis through Inactivation of Notch-1 and Nuclear Factor-κB Signaling

doi: 10.1158/0008-5472.can-07-2803

Figure Lengend Snippet: Figure 5. A, nuclear proteins from siRNA- and cDNA-transfected cells were subjected to analysis for NF-nB DNA-binding activity as measured by EMSA. Left, down-regulation of PDGF-D inhibited NF-nB DNA-binding activity compared with control; middle, PDGF-D cDNA transfection caused activation of NF-nB DNA-binding activity in all three cell lines tested; right, NF-nB supershift analyses. EMSA experiments were done by additional 30-min incubations with polyclonal supershift antibodies against p65 before the addition of labeled probe. Lane 1, nonspecific antibody (anti-cyclin D1); lane 2, p65 antibody. B, left, Western blot analysis showed that PDGF-D cDNA transfection increased the expression of MMP-9 and VEGF; right, real-time RT-PCR showed that PDGF-D cDNA increased the expression of MMP-9 genes at mRNA level in pancreatic cancer cells. C, left, PDGF-D cDNA transfection increased the activity of MMP-9 in pancreatic cancer cells; right, PDGF-D cDNA transfection increased the secreted levels of VEGF in pancreatic cancer cells. *, P < 0.05; **, P < 0.01, relative to control.

Article Snippet: PDGF-D siRNA and control siRNA were obtained from Santa Cruz Biotechnology.

Techniques: Transfection, Binding Assay, Activity Assay, Control, Activation Assay, Labeling, Western Blot, Expressing, Quantitative RT-PCR