Fig 2: Focal ischemia induces upregulation of PKD2 protein and activity in the peri-infarct regions after tMCAO. A. PKD2 was upregulated in ipsilateral tissues. QPCR analysis was used to identify Prkd1–3 mRNA expression in contralateral and ipsilateral sides of mouse brain at 24 h reperfusion after 1 h MCAO. Data are Mean ± SEM of triplicate determinations from a representative experiment of three such similar experiments. C, cortex; H, hippocampus; S, striatum; IC, ischemic core, PI, peri-infarct; Cont, contralateral; Ipsi, Ipsilateral. B. WT mouse brain tissue subjected to 24 h reperfusion after 1 h MCAO was dissected and subjected to Western blot analysis followed by immunoblotting for several targets. Representative images from three experiments are shown. C. The activity of PKD increased in the ipsilateral ischemic tissue (cortex and PI) compared to the control tissue in the contralateral cortex. Quantification of p-S744/748-PKD in the Western blots shown in “B” and performed by densitometry analysis. GAPDH was blotted as the loading control. D. The endogenous expression and activity of PKD2 in the peri-infarct area of WT and KI subjected to 1 h MCAO followed by 24 h reperfusion. Mouse brain sections from WT and KI mice were stained for PKD2 and p-S876-PKD2 by IF. Scale bar = 20 μM. Representative images from three experiments are shown. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.

Fig 3: Prostate cancer cells-derived PKD2/3 promote chemotactic migration of mast cells and endothelial cells tube formation. a Conditional medium (CM) from PC-3 M or DU145 cells transiently transfected with si-CTL, si-PKD2, si-PKD3 was used to be chemoattractant for P815 mast cell migration by transwell assays. ***p < 0.001, **p < 0.01 versus si-CTL by one-way ANOVA. b Silencing of PKD2 and PKD3 verification by Western blotting (left panel). c VEGF mRNA level was assessed by qPCR in P815 mast cells induced by conditional medium from DU145 cells transfected with siRNA of PKD2 or /and PKD3 (right panel). *p < 0.05, **p < 0.01 versus si-CTL by one-way ANOVA. d Knockdown efficiency were verified by Western blot in PC-3 M(up) or P815 mast cells(bottom). Cells were transiently transfected with siRNA of indicated genes using lipofectmine3000. e HUVEC cells were treated with conditional medium from co-cultured of P815 mast cells transfected with si-CTL or si-VEGF and PC-3 M cells transfected with si-CTL, si-PKD2, si-PKD3 or si-PKD2 + 3. Tube formation of HUVEC cells were visualized by phase contrast inverted microscope (100×). f Tube formation was assessed the number of nodes per image was quantified and analyzed by two way ANOVA, **p < 0.01

Fig 4: 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

Fig 5: 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