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: Scientific Reports

Article Title: Single-nucleus transcriptomic profiling of the diaphragm during mechanical ventilation

doi: 10.1038/s41598-024-82530-4

Figure Lengend Snippet: Differentially expressed genes and Kyoto Encyclopedia of Genes and Genomes analysis. Volcano plots showing the top twenty up- or downregulated genes for each cell type. The red dots represent upregulated genes, and the blue dots represent downregulated genes. ( P value < 0.05, and |log2foldchange| > 0.58). Bubble plot indicating the top enriched pathways for each cell type based on KEGGpathway enrichment analysis of differentially expressed genes( www.kegg.jp/kegg/kegg1.html ). The sizes of the dots represent the number of genes included in each pathway. The colour gradient of dots represents the adjusted P values of each enriched pathway. The genes Pfkfb3 , Tbc1d1 and the insulin signaling pathway; Pdgfd and the PI3K-Akt signaling pathway; Cxcr2 and PLD, Rap1 signaling pathways; Ccl21 and chemical carcinogenesis-reactive oxygen species signaling pathway; Mef2c and the calcium signaling pathway; Negr1 and leukocyte transendothelial migration signaling pathway are labelled by the black and red bars in Panels a , b , c , d , e , and f .

Article Snippet: After the semidry blotting procedure (50 min, 90 V), the membrane was incubated for 1 h at room temperature (RT) in 5% BSA blocking solution, followed by overnight incubation on a shaker at 4 °C with primary antibodies against PFKFB3 (bs-3528R, Boster Biological Technology Co., Ltd., Wuhan, China), PDGFD (bs-24572R, Boster), CXCR2 (bs-1629R, Boster), NEGR1 (bs-11095R, Boster), SEMA3A (bs-10468R, Boster), MEF2C (bs-4130R, Boster) and β-actin (66009-1-Ig, Proteintech, Wuhan, China) as a loading control.

Techniques: Protein-Protein interactions, Migration

Journal: Scientific Reports

Article Title: Single-nucleus transcriptomic profiling of the diaphragm during mechanical ventilation

doi: 10.1038/s41598-024-82530-4

Figure Lengend Snippet: High-dimensional weighted gene coexpression network analysis (hdWGCNA). a The top left panel depicts the soft power threshold for choosing a scale-free topology model. The average connectivity of the topological network was most stable at the lowest soft threshold of 6. b Dynamic Tree Cut algorithm for gene clustering. Each leaf on the tree represents a gene, and the colour at the bottom indicates the assignment to a specific coexpression module. c Bubble plots displaying the scores obtained for eight modules in eight cell subtypes. d snRNA-seq UMAP coloured by module eigengene (ME) for eight coexpression modules. e The eight modules’ values of -log10 (Adj P value) on the Y-axis versus average log2 (fold change) on the X-axis are shown for each cell type, including endothelial cells, fibroblast 1, fibroblast 2, myocytes, neurocytes, pericytes, satellite cells and smooth muscle cells. The larger the value is, the greater the fit between the coloured module and the specific cell type. The plot shows that fibroblast 1 features a brown module, fibroblast 2 features a green module, endothelial cells feature a yellow module, and myocyte cells feature a turquoise module. f UMAP plot of the gene coexpression network with eight coloured modules and the top two hub genes labelled in each module. Nodes are coloured according to coexpression module assignment. g Hub genes in each module were identified by eigengene-based connectivity (KME). The following five hub genes associated with fibrosis were identified: Pfkfb3 and Tbc1d1 in the brown module, Pdgfd in the green module, Sema3a in the yellow module and Ryr3 in the turquoise module.

Article Snippet: After the semidry blotting procedure (50 min, 90 V), the membrane was incubated for 1 h at room temperature (RT) in 5% BSA blocking solution, followed by overnight incubation on a shaker at 4 °C with primary antibodies against PFKFB3 (bs-3528R, Boster Biological Technology Co., Ltd., Wuhan, China), PDGFD (bs-24572R, Boster), CXCR2 (bs-1629R, Boster), NEGR1 (bs-11095R, Boster), SEMA3A (bs-10468R, Boster), MEF2C (bs-4130R, Boster) and β-actin (66009-1-Ig, Proteintech, Wuhan, China) as a loading control.

Techniques:

Journal: Scientific Reports

Article Title: Single-nucleus transcriptomic profiling of the diaphragm during mechanical ventilation

doi: 10.1038/s41598-024-82530-4

Figure Lengend Snippet: Protein-protein interaction network (PPI network).We constructed nine PPI networks displaying protein-protein interactions among the related genes. The nodes represent proteins, and the edges represent the interaction strength between two proteins. The proteins PFKFB3 ( a ), PDGFD ( b ), CXCR2 ( c ), CCL21 ( d ), SEMA3A ( e ), RYR3 ( f ), MEF2C ( g ), NEGR1( h ) and TBC1D1 ( i ) which are located at the hub of the interaction network, are responsible for diaphragm fibrosis and atrophy.

Article Snippet: After the semidry blotting procedure (50 min, 90 V), the membrane was incubated for 1 h at room temperature (RT) in 5% BSA blocking solution, followed by overnight incubation on a shaker at 4 °C with primary antibodies against PFKFB3 (bs-3528R, Boster Biological Technology Co., Ltd., Wuhan, China), PDGFD (bs-24572R, Boster), CXCR2 (bs-1629R, Boster), NEGR1 (bs-11095R, Boster), SEMA3A (bs-10468R, Boster), MEF2C (bs-4130R, Boster) and β-actin (66009-1-Ig, Proteintech, Wuhan, China) as a loading control.

Techniques: Construct, Protein-Protein interactions

Journal: Scientific Reports

Article Title: Single-nucleus transcriptomic profiling of the diaphragm during mechanical ventilation

doi: 10.1038/s41598-024-82530-4

Figure Lengend Snippet: Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. qRT-PCR and Western blotting showing the expression of selected genes and corresponding proteins in the mechanically ventilated diaphragm and control ones. The bar graphs show the quantification of the mRNA expression of Pfkfb3 ( a ), Pdgfd ( b ), Cxcr2 ( c ), Negr1 ( d ), Sema3a ( e ), and Mef2c ( f ) normalized to that of GAPDH. Asterisks indicate significant differences ( n = 3 in each group) (* P < 0.05). Western blotting analysis of the protein expression of PFKFB3 ( g ), PDGFD ( h ), CXCR2 ( i ), NEGR1 ( j ), SEMA3A ( k ), and MEF2C ( l ) normalized to that of β-actin as a loading control.

Article Snippet: After the semidry blotting procedure (50 min, 90 V), the membrane was incubated for 1 h at room temperature (RT) in 5% BSA blocking solution, followed by overnight incubation on a shaker at 4 °C with primary antibodies against PFKFB3 (bs-3528R, Boster Biological Technology Co., Ltd., Wuhan, China), PDGFD (bs-24572R, Boster), CXCR2 (bs-1629R, Boster), NEGR1 (bs-11095R, Boster), SEMA3A (bs-10468R, Boster), MEF2C (bs-4130R, Boster) and β-actin (66009-1-Ig, Proteintech, Wuhan, China) as a loading control.

Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Expressing, Control