Fig 2: MDM2 overexpression downregulated the translation level of PTN while MDM2 silencing did the opposite. A, the factor which mediates the PTN ubiquitination was analyzed by UbiBrowser (http://ubibrowser.ncpsb.org/). B and C, after MDM2 overexpression or silencing, the transcription and translation levels of PTN were evaluated through qRT-PCR (B) and Western blot (C). (***p < 0.001, versus NC; ###p < 0.001, versus sh-NC). MDM2, murine double minute 2; NC, negative control; OE, overexpressed; PTN, Pleiotrophin; qRT-PCR, quantitative RT-PCR; Sh, short hairpin.

Fig 3: Triad1 inhibited the MDM2-mediated PTN ubiquitinationin HEK-293 cells.A, PTN ubiquitination in HEK-293 cells when Flag-PTN and His-ubiquitin were cotransfected with HA-MDM2 or not was detected by Co-IP. B, in the presence or absence of MG132, PTN ubiquitination in HEK-293 cells when Flag-PTN and His-ubiquitin were cotransfected with HA-MDM2 or not was determined by Co-IP. C and D, the protein expressions of PTN and Triad1 in astrocytes transfected with plasmids overexpressing MDM2 and Triad1 were detected by Western blot. E, in the presence or absence of MG132, PTN ubiquitination in HEK-293 cells when Flag-PTN, His-ubiquitin, and HA-MDM2 were cotransfected with Myc-Triad1 or not was detected by Co-IP. (***p < 0.001, versus NC; ###p < 0.001, versus OE-MDM2; ˆˆp < 0.01, versus OE-Triad1). Co-IP, coimmunoprecipitation; MDM2, murine double minute 2; NC, negative control; OE, overexpressed; PTN, pleiotrophin; Triad1, two RING fingers and DRIL 1.

Fig 4: The expressions of Triad1, MDM2, and PTN were increased in neurons and in astrocytes following SCI. A, coexpression of Triad1 with NeuN. B, coexpression of Triad1 with GFAP. C, coexpression of PTN with NeuN. D, coexpression of PTN with GFAP. E, coexpression of MDM2 with NeuN. F, coexpression of MDM2 with GFAP. G, relative fluorescence intensity in the sham and SCI-1 day injury groups. Magnification: ×200; scale bars: 50 µm. (**P < 0.01, ***P < 0.001 versus sham). (SCI, spinal cord injury; Triad1, two RING fingers and DRIL 1; PTN, Pleiotrophin; MDM2: murine double minute 2)

Fig 5: Triad1, PTN and MDM2 interacted with each other in SCI rats. A, Triad1-positive cells, MDM2-positive cells, and PTN-positive cells in spinal cord tissues at 1 day after surgery were detected through immunohistochemistry. Magnification: ×200; scale bars: 100 µm. B and C, Co-IP was performed to explore the association between MDM2, PTN, and Triad1 in spinal cord tissues at 1 day after surgery. Co-IP, coimmunoprecipitation; MDM2, murine double minute 2; PTN, pleiotrophin; SCI, spinal cord injury; Triad1, two RING fingers and DRIL 1.