Journal: STAR Protocols

Article Title: Generation of hematopoietic lineage cell-specific chimeric mice using retrovirus-transduced fetal liver cells

doi: 10.1016/j.xpro.2022.101526

Figure Lengend Snippet:

Article Snippet: Mouse SCF, research grade , Miltenyi Biotec , 130-101-741.

Techniques: Virus, Recombinant, Marker, Modification, Saline, Transfection, Western Blot, Plasmid Preparation, Purification, Gel Extraction, Retroviral, Sequencing, Software, Flow Cytometry

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Protein kinase Ds promote tumor angiogenesis through mast cell recruitment and expression of angiogenic factors in prostate cancer microenvironment

doi: 10.1186/s13046-019-1118-y

Figure Lengend Snippet: PKD2/3 enhance MCs migration through upregulation of SCF, CCL5 and CCL11 in prostate cancer cellsmRNA and the protein level of the scf , ccl5 and ccl11 were analyzed by real-time qPCR and ELISA in DU145 ( a ) and PC-3 M ( b ) cells transfected with siRNA of PKD2, PKD3. Data representing the means ± S.D. of three independent experiments was analyzed by one-way ANOVA for significance versus si-CTL. ***p < 0.001, **p < 0.01, *p < 0.05 versus si-CTL. c Knockdown efficiency of PKD2 and PKD3 in prostate cancer cells was verified by Western blotting. d DU145 cells were transfected with siRNA of PKD2, PKD3, the Conditional medium (CM) was collected to measure migration of P815 cells in response to SCF, CCL5, and CCL11 treatment by transwell assay. e Quantification were analyzed from data in D. ***p < 0.001 versus si-CTL by One-ANOVA tests

Article Snippet: Quantitative measurement of cytokines, including stem cell factor (SCF), Chemokine ligand 5 (CCL5), C-C motif chemokine 11(CCL11) and vascular endothelial growth factor (VEGF) secreted into conditioned medium was determined using ELISAs, according to the manufacturer’s protocol (BOSTER).

Techniques: Migration, Enzyme-linked Immunosorbent Assay, Transfection, Knockdown, Western Blot, Transwell Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Protein kinase Ds promote tumor angiogenesis through mast cell recruitment and expression of angiogenic factors in prostate cancer microenvironment

doi: 10.1186/s13046-019-1118-y

Figure Lengend Snippet: PKD2 and PKD3 promote SCF, CCL5 and CCL11 expression through Erk1/2 signaling pathways. a-b Interaction of PKD2 or PKD3 with Erk1/2 was performed by co-IP assay in PC-3 M or DU145 prostate cancer cells. c-d DU145 cells ( c ) or PC-3 M cells ( d ) were transfected as indicated and treated with 100 nM PMA, phosphorylation and protein expression were detected by western blotting. e Overexpression efficiency of PKD2 and PKD3 in prostate cancer cells was verified by Western blotting. f ELISA were applied to measure SCF in conditional medium from DU145 cell transfected with GFP, GFP-PKD2, and GFP-PKD3 in present with or without Erk inhibitor PD98059(PD) treatment. g Real-time PCR was performed to analyze ccl5 expression in DU145 transfected with GFP, GFP-PKD2 and GFP-PKD3 plasmids followed by treatment with or without Erk inhibitor PD98059

Article Snippet: Quantitative measurement of cytokines, including stem cell factor (SCF), Chemokine ligand 5 (CCL5), C-C motif chemokine 11(CCL11) and vascular endothelial growth factor (VEGF) secreted into conditioned medium was determined using ELISAs, according to the manufacturer’s protocol (BOSTER).

Techniques: Expressing, Protein-Protein interactions, Co-Immunoprecipitation Assay, Transfection, Phospho-proteomics, Western Blot, Over Expression, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Protein kinase Ds promote tumor angiogenesis through mast cell recruitment and expression of angiogenic factors in prostate cancer microenvironment

doi: 10.1186/s13046-019-1118-y

Figure Lengend Snippet: PKD2 and PKD3 are required for SCF, CCL5, and CCL11 transcription. a Analysis of the binding site of p65(highlight in red) and AP1(highlight in blue) in the promoters of scf, ccl5 and ccl11 using UCSC software online. b Western blotting was used to ensure the knockdown effect. ChIP analysis of the binding of c-Jun ( c ), c-Fos (d) and NF-κB ( e ) to the scf, ccl5 and ccl11 gene promoter in PC-3 M cells depleted with siRNA of PKD2, PKD3. Student’s t-test , *p < 0.05, **p < 0.01 and ***p < 0.001 ( n = 3). Error bars indicate mean ± S.D.

Article Snippet: Quantitative measurement of cytokines, including stem cell factor (SCF), Chemokine ligand 5 (CCL5), C-C motif chemokine 11(CCL11) and vascular endothelial growth factor (VEGF) secreted into conditioned medium was determined using ELISAs, according to the manufacturer’s protocol (BOSTER).

Techniques: Binding Assay, Software, Western Blot, Knockdown

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Protein kinase Ds promote tumor angiogenesis through mast cell recruitment and expression of angiogenic factors in prostate cancer microenvironment

doi: 10.1186/s13046-019-1118-y

Figure Lengend Snippet: CRT0066101 reduces MCs recruitment and tumor angiogenesis in vivo. a Experimental setting. b C57BL6 mice bearing RM1 tumors were administered a daily vehicle [control group; 5% ( w / v ) dextrose] or CRT0066101 at 20 μM and 40 μM for 2 weeks (4 mice per group), then excised tumor images. c Tumor volume were represented at indicated day. d Immunohistochemistry staining for phospho-PKD, microvessel density (stained with CD31), and mast cells (stained with c-Kit). Representative image were shown in 400X under the microscope (Left panel). Quantification of the indicated parameter was analyzed among groups after treatment with CRT0066101 (Right panel). e Schematic model of the mechanistic role of PKD2 and PKD3 in tumor angiogenesis by regulating SCF-, CCL5-, and CCL11-mediated mast cell recruitment

Article Snippet: Quantitative measurement of cytokines, including stem cell factor (SCF), Chemokine ligand 5 (CCL5), C-C motif chemokine 11(CCL11) and vascular endothelial growth factor (VEGF) secreted into conditioned medium was determined using ELISAs, according to the manufacturer’s protocol (BOSTER).

Techniques: In Vivo, Control, Immunohistochemistry, Staining, Microscopy