Fig 2: A schematic summary of the involvement of PKC-? and PKC-? in melanoma progression. Upon extra cellular stimulation with TNF-a and TGFß, PKC-? activates Par6, which leads to the degradation of RhoA and stimulates EMT by changing the cell integrity, loss of E-cadherin and gain of Vimentin. During this process, cadherin junctions will be destabilized as a result of loss of E-cadherin and ß-catenin will be translocated to nucleus to upregulate the production of some proteins such as CD44 which further stimulate migration and EMT. Importantly PKC-? tightly binds to Vimentin to activate them by phosphorylation and this activated Vimentin changes the cell polarity to maintain the mesenchymal phenotype. Activated Vimentin can also induce the phosphorylation of PTEN leading to inactivation of inhibitory action of PTEN on PIP3 [40]. This may result in activation of AKT through PIP3 and activated AKT pathway leading to cell survival, rapid proliferation and differentiation which are critical parts of melanoma progression. AKT could indirectly stimulate ß-catenin translocation and activate NF-?B pathway in which PKC-? is known to play a stimulatory role on IKK-a/ß. It is reported that activated NF-?B can inhibit PTEN [35].

Fig 3: NCAPG enhances the proliferation of HCC cells and affects the PI3K-AKT pathway by affecting the expression of PTEN. A Western blot analysis showing the levels of PTEN, AKT, p-PTEN and p-AKT when NCAPG was overexpressed. Quantitative analysis showed the same pattern. B Western blot analysis showing the levels of PTEN, AKT, p-PTEN and p-AKT when NCAPG was knocked down. Quantitative analysis showed the same pattern. C When MK2206 was added to NCAPG-overexpressing cells, the levels of AKT and p-AKT were reversed. D and E The cell proliferation ability of HCCLM3 and MHCC97H cells, as evaluated by EdU incorporation and CCK-8 assays, was attenuated when NCAPG was upregulated with MK2206. The values indicate the means ± SDs of three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001; independent Student’s t test)

Fig 4: The effects of i.t. injection of Ad-PTEN on PTEN in the dorsal lumbar spinal cord. Fourteen days after i.t.; spinal cord sections (10 µm) are from i.t. injection of vehicle (A), Ad-GFP (B), and Ad-PTEN (C) groups. Immunostaining images of the spinal cord show cells labeled with PTEN (red). Quantification of PTEN (D) immunoreactivity in the ipsilateral dorsal horn of the lumbar spinal gray matter compared with vehicle group. Injection (i.t.) of Ad-PTEN significantly upregulated spinal PTEN immunoreactivity. Each bar in (D) represents the mean ± SEM with six rats per group. Ad-GFP, adenovirus-mediated green fluorescent protein; Ad-PTEN, adenovirus-mediated phosphatase and tensin homolog deleted from chromosome 10. Confocal double-immunofluorescent staining of PTEN (red) with NeuN (neuronal-specific marker; (E), green), GFAP (astrocyte specific marker; (F), green), and OX-42 (microglial specific marker; (G), green) in the dorsal horn region of the lumbar spinal cord of the i.t. Ad-PTEN group. The merged images of (E), (F), and (G) (yellow; white arrow) indicate co-localization of PTEN with NeuN, GFAP, and OX-42 immunoreactive cells in the spinal cord, respectively. The confocal results show that spinal PTEN was primarily co-localized with astrocytes in i.t. Ad-PTEN group. Scale bars are 200 µm for panels A-C and 50 µm for panels E-G. *P < 0.05 compared with the vehicle group.

Fig 5: Effects of the time course of i.t. Ad-PTEN on the spinal PTEN and phospho-mTOR levels in CCI rats. (A) Western blots for PTEN, phospho-mTOR, and ß-actin proteins from sham-operated plus i.t. vehicle 7 days post injury, CCI plus i.t. Ad-GFP 7 days post injury, CCI plus i.t. Ad-PTEN 7 days post injury, sham-operated plus i.t. vehicle 14 days post injury, CCI plus i.t. Ad-GFP 14 days post injury, and CCI plus i.t. Ad-PTEN 14 days post injury. (B) Relative density of the immunoblot of PTEN. (C) Relative density of the immunoblot of phospho-mTOR. Relative band intensities of PTEN and phospho-mTOR were quantified by densitometry and indicated as the percent change relative to that for the sham-operated 7 days post injury group (100%). Western blotting revealed that PTEN and phospho-mTOR protein expression were detectable in the sham-operated group, but CCI-induced downregulation of PTEN and upregulation of phospho-mTOR protein on day 7 and day 14 after CCI surgery were both attenuated by i.t. Ad-PTEN. Western blotting of ß-actin was performed to verify that equivalent amounts of protein were loaded in each lane. Each bar in (B) and (C) represents the mean ± SEM with three rats per group. *P < 0.05 compared with sham-operated 7 days post injury group. # P < 0.05 between CCI plus i.t. Ad-PTEN group compared with the same time points in CCI plus i.t. Ad-GFP group. Ad-GFP, adenovirus-mediated green fluorescent protein; Ad-PTEN, adenovirus-mediated phosphatase and tensin homolog deleted from chromosome 10; CCI, chronic constriction injury; p-mTOR, phosphorylated mammalian target of rapamycin.