pdgf aa  (R&D Systems)

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: MDA-9/Syntenin in the tumor and microenvironment defines prostate cancer bone metastasis.

doi: 10.1073/pnas.2307094120

Figure Lengend Snippet: Fig. 6. Tumor cell–derived PDGF-AA induces CXCL5 expression in HS5 cells. (A) Normalized (based on equal amounts of total protein) conditioned media, isolated from parental and knockout variants, were subjected to protein-based growth factor arrays as described by Ray Biotech. Heatmap is presented. Densitometry analyses (n = 2, for corresponding proteins) were performed for each growth factor, and the data are presented only for three. (B) HS5 cells were treated for the indicated times with PDGF-AA, and CXCL5 protein expression was determined. (C) HS5 cells were transfected with a 1.5-kb CXCL5-Prom using a standard transfection protocol. Twelve hours posttransfection, cells were treated with PDGF-AA for an additional 12 h. Luciferase activity was measured and presented after normalizing with Renilla luciferase. (D) PC-3ML-derived conditioned media were incubated with control IgG or anti-PDGF-AA before treatment of HS5 cells for 12 h, and CXCL5 levels were measured in media. (E) Expression of PDGF-AA was determined in the indicated tumor cells and corresponding mda-9 knockout clones. (F) Cells were infected with an Adenovirus expressing control or mda-9 overexpressing construct for 24 h, and PDGF-AA expression in media was determined using ELISA. (G) HS5 cells were treated with PDGF-AA and analyzed for expression of the indicated proteins. (H) Tumor cells were cultured in the presence or absence of IKK2i for 12 h and analyzed for expression of the indicated proteins. Different letters in two variables are statistically significant (P < 0.05). (I) MDA-9 was knocked-into PC-3MLmda-9 KO cells and cultured for an additional 12 h in the presence or absence of IKK2i. PDGF-AA was determined using ELISA. *=P < 0.05.

Article Snippet: Neutralizing antibodies for CXCL5 and PDGF- AA were obtained from R&D Systems.

Techniques: Derivative Assay, Expressing, Isolation, Knock-Out, Transfection, Luciferase, Activity Assay, Incubation, Control, Clone Assay, Infection, Construct, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: MDA-9/Syntenin in the tumor and microenvironment defines prostate cancer bone metastasis.

doi: 10.1073/pnas.2307094120

Figure Lengend Snippet: Fig. 7. MDA-9 expression in HS5 regulates PDGF/PDGFRα signaling. (A) mda-9 expression was blunted, and 24 h postinfection, HS5 cells were stimulated with PDGF-AA for an additional 12 h. mRNA for CXCL5 expression was analyzed using qPCR. Different letters in two variables are statistically significant (P < 0.05). (B) HS5 cells were incubated with media supplemented with the indicated tumor cell–derived conditioned media for 12 h, and western blotting was done for phosho-PDGFRα and total PDGFR. (C) HS5 cells were incubated with media supplemented with the indicated tumor cell–derived conditioned media and PDGF- AA for 12 h, and western blotting was done for the indicated proteins. (D) BM-MSCs from WT and mda-9−/− mice were stimulated with mouse PDGF-AA, and CXCL5 expression was determined in the culture media. (E) phospho-PDGFR expression was analyzed in paraffin sections (tumor-bearing bone from the animal experiment, described in Fig. 2A). Different letters in two variables are statistically significant (P < 0.05).

Article Snippet: Neutralizing antibodies for CXCL5 and PDGF- AA were obtained from R&D Systems.

Techniques: Expressing, Incubation, Derivative Assay, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: MDA-9/Syntenin in the tumor and microenvironment defines prostate cancer bone metastasis.

doi: 10.1073/pnas.2307094120

Figure Lengend Snippet: Fig. 8. CXCL5 is expressed in both stroma and tumor cells. (A) Paraffin-embedded section from a patient with metastatic PC bone metastasis was immunostained with anti-CXCL5, and the expression in the stroma and tumor compartment is presented in the Inset. (B) H-Score was determined by an anatomic pathologist based on staining intensity, and values are presented. (C) PC3-ML cells were stained with CytoTrack™ Red and cocultured with of HS5 cells. After 24 h, cells were stained with CXCL5-FITC and analyzed by FACS [the left panel represents double staining (tumor cell-Red and CXCL5-FITC)]; the middle panel is for only CXCL5- FITC (only HS5). The right panel represents conditioned media that were collected from the above indicated PC3-ML and HS5 cocultured cells, and secretion of CXCL5 was measured by ELISA. (D) Human serum samples from the indicated patient groups were analyzed for PDGF-AA and CXCL5 (n = 20). Different letters in two variables are statistically significant (P < 0.05). *Statistically significant. **P < 0.001.

Article Snippet: Neutralizing antibodies for CXCL5 and PDGF- AA were obtained from R&D Systems.

Techniques: Expressing, Staining, Double Staining, Enzyme-linked Immunosorbent Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: MDA-9/Syntenin in the tumor and microenvironment defines prostate cancer bone metastasis.

doi: 10.1073/pnas.2307094120

Figure Lengend Snippet: Fig. 9. Schematic of the proposed role of MDA-9 in BM- MSCs in creating a favorable environment for metastatic outgrowth through secretion of CXCL5 in response to tumor cell–derived growth factor, PDGF-AA.

Article Snippet: Neutralizing antibodies for CXCL5 and PDGF- AA were obtained from R&D Systems.

Techniques: Derivative Assay

Journal: Biochemical and biophysical research communications

Article Title: The role of bone-derived PDGF-AA in age-related pancreatic β cell proliferation and function.

doi: 10.1016/j.bbrc.2019.12.057

Figure Lengend Snippet: Fig. 3. Conditioned medium from osteoblast lineage cells induces b cells proliferation and function via PDGF-AA. We cultured rat insulinoma INS1 cells using standard medium(CON) or osteoblast-lineage-cells conditioned medium (OB CM) with or without addition of PDGF-AAeneutralizing antibody(details in materials and methods). Images of senescence-associated b-galactosidase (SA-bGal) staining (blue) in (A). Immunofluorescence staining of the cells were performed using antibodies against p16INK4a (B), Ki67 (C) and Insulin (D), respectively. DAPI stains nuclei blue. The expressions of b cell proliferation-associated genes Rb(G) and Foxo1(H) or b cell specific genes Pdx1(I) and Ins1(J) were detected by qPCR. Data are represented as mean ± s.e.m. *p < 0.05 as determined by one-way ANOVA.

Article Snippet: The cells were allowed to grow for 2e3 days and serum-containing conditioned mediumwas harvested from the osteoblast lineage cells and stored at 80 C. In some experiments, we added the neutralizing antibodies for PDGF-AA (R&D).

Techniques: Cell Culture, Staining

Journal: Biochemical and biophysical research communications

Article Title: The role of bone-derived PDGF-AA in age-related pancreatic β cell proliferation and function.

doi: 10.1016/j.bbrc.2019.12.057

Figure Lengend Snippet: Fig. 4. Transplantation of osteoblast lineage cells increases b cells proliferation and insulin secretion in aging mice. Immunofluorescence staining of femur metaphysis sections was performed using antibodies against Osx (red) (A). Serum PDGF-AA level was measured in 6-months-old adult mice and in 22-months-old aging mice with statistics in (B). The fasting levels of serum insulin in (C) and blood glucose in (D). Immunofluorescence staining of PDGFr-a activation in (E). The expressions of p16 and Ki67 in pancreas tissue were detected by qPCR in (F) and (G), respectively. Staining of SA-bGal in (H) and immunofluorescence staining of Insulin in (I). (J) An IPGTT was performed and serum glucose level was measured at designated timepoints after glucose loading. Data are represented as mean ± s.e.m. *p < 0.05 as determined by Student’s t-tests.

Article Snippet: The cells were allowed to grow for 2e3 days and serum-containing conditioned mediumwas harvested from the osteoblast lineage cells and stored at 80 C. In some experiments, we added the neutralizing antibodies for PDGF-AA (R&D).

Techniques: Transplantation Assay, Staining, Activation Assay