Figure 5. NNMT silencing decreases STK activation by methylating PP2A-C.

(A) Immunohistochemical staining of tumors harvested from mice injected with KD and OE cells. The tissue sections were stained with H&E and Akt, P-Akt, p44/42 MAPK, and P-p44/42 MAPK antibodies for immunodetection.

(B) Western blot analysis of the change in PP2A activation, corresponding LCMT1 expression levels, and activation of STKs in the KD and OE cell lines (Ratio = expression level normalized to β-Actin) [Ratio* = Expression level of (Me-PP2A-C:β-Actin):(PP2A:β-Actin)]. The control group of wild type, knockdown, and overexpression is shown on the right.

(C) Basal level of PP2A activation in the NNMT isogenic cell lines (Ratio = Expression level of (Me-PP2A-C:β-Actin):(PP2A:β-Actin)).

(D) The in vitro activity of PP2A in the KD and OE cell lines.

(E) LC-MS/MS was used to determine the concentrations of the substrates and products involved in the NNMT enzymatic reaction, along with the methylation potential (MP) for the GBM cell lines tested. The graph to the right represents the relative MP of the KD and OE cell lines.

(F) Western blots showing an increased methylation of PP2A-C and a consequent inhibition of P-Akt and P-p44/42 MAPK in U87-KD₃₃₀ and MGH8-KD cells.