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.