Fig 2: PTN-expressing myoblast implantation in normal and ischemic leg muscle.A,B) PTN staining and ß-gal staining of a single section from non-ischemic mouse leg muscle implanted with myoblasts expressing both PTN and LacZ genes confirms that PTN is expressed in muscle fibers after myoblast fusion. C) The merged image shows triple staining for capillaries, smooth muscle cells (SMA = smooth muscle actin), and ß-gal of a similar section. While some visible increases in vascularity occurred at some implantation sites relative to the surrounding tissue as pictured here, this happened in control LacZ implantation sites as well. Bar = 50 µm. D,E) No significant differences in arteriole density and capillary index were observed in non-ischemic muscles injected with PTN myoblasts versus LacZ myoblasts (n = 5). F,G) Representative laser Doppler images at 14 days showing a significant increment in skin perfusion at the feet after PTN myoblast treatment, in comparison with the control LacZ myoblast group (n = 12 for PTN, n = 10 for LacZ). H) Quantification of skin perfusion at feet of ischemic limbs based on laser Doppler measurements. *p<0.05. I) A significantly higher number of arterioles were detected in ischemic leg muscle implanted with PTN myoblasts than with LacZ myoblasts (n = 4), *p<0.05. J) No significant difference was observed in capillary index (n = 4). Smooth muscle actin (SMA) staining of arterioles (K,L) and CD31 staining of capillaries (M,N) in representative sections from ischemic leg muscle implanted with PTN myoblasts vs. LacZ myoblasts. Scale bar = 25 µm.

Fig 3: No evidence of tumorigenesis from full-length PTN expression in myoblasts.A) When PTN myoblasts and lacZ myoblasts were plated at equal densities and grown past confluence, the cells proliferated and declined at equal rates regardless of PTN expression. B) There was no difference between the cell types in percent viability after contact inhibition. C) The positive control MDA-MB-231 breast cancer cell line induced subcutaneous tumors at 3/6 implantation sites after 4 weeks (black arrows), whereas implantation of PTN myoblasts resulted in no detectable tumor formation (0/6; white arrows) in this experiment, and 0/5 after 10 weeks in a separate experiment (not shown). 1×105 cells were injected in 10 µl PBS for anterior injections and 10 µl Matrigel for posterior injections. Animals are shown 4 weeks post injection. Tumor #1 = 1×0.8×0.5 cm; tumor #2 = 0.3×0.3×0.2 cm; tumor #3 = 0.7×0.9×0.5 cm.

Fig 4: NRP-1 and PTN interaction activates MAPK, PI3K, and FAK pathways. (A) PC3 cells were transiently transfected with NRP-1 siRNA or with control siRNA. NRP-1 protein level was evaluated by IB. (B) NRP-1 knockdown reduced PTN-induced signaling. Transfected PC3 cells were serum starved overnight and then incubated with PTN (100 ng/ml) for 15 minutes at 37°C. Cell lysates were analyzed by IB with anti–phospho-MAPK (ERK1/2), phospho-Akt, and phospho-FAK tyr925 as well as anti-total MAPK, Akt, and FAK. Ctl: control. The phospho-protein levels obtained by scanning densitometry were normalized to total protein, respectively. Results shown are representative of three independent experiments. Bars, SEM. **P < .01; ***P < .001 when compared with respective controls (-, Ctl). (C) Transfected PC3 cells were serum starved overnight and then incubated with VEGF A165 (50 ng/ml) for 15 minutes at 37°C. (D) Transfected PC3 cells were serum starved overnight and then incubated with FGF2 (50 ng/ml) for 15 minutes at 37°C. Immunoblotting was done as described above.

Fig 5: Pleiotrophin (PTN), midkine (MK), and receptor protein tyrosine phosphatase ß/? (RPTPß/?) protein expression are altered by acute and chronic morphine administration and during morphine withdrawal in the nucleus accumbens (NAc). Over a 7 day period, control (pla) and morphine (mor)-dependent rats received saline (sal), morphine (mor; 20mg/kg i.p.), or naloxone (nx; 1mg/kg s.c.) on day 7 and were sacrificed 2h later. Semi-quantitative analysis and representative immunoblots of PTN (A, B), MK (C, D), and RPTPß/? (E, F) proteins in the NAc were isolated from rats receiving the above treatments. Each bar corresponds to the mean ± standard error of the mean. Values are expressed as a % of controls. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. pla+sal; + p < 0.01, ++ p < 0.001 vs. pla+mor; # p < 0.05, ## p < 0.01 vs. pla+nx; & p < 0.05, && p < 0.01, &&& p < 0.001 vs. mor+sal