RT-qPCR and immunoblotting were used to assess pAKT-2, AKT-2 and MYCN mRNA and protein expression in control (shCTL), SHMT2 silencing (shSHMT2) and SHMT2 overexpression (pCo-SHMT2) cell lines. (A) SHMT2 silencing decreased AKT-2 mRNA expression by 1.2-fold and SHMT2 overexpression increased AKT-2 mRNA expression by 1.6-fold in SK-N-AS cells. (B) SHMT2 silencing decreased AKT-2 expression by 2.6-fold in BE(2)-C cells and SHMT2 overexpression increased AKT-2 mRNA expression by 1.3-fold. MYCN mRNA expression was increased by 1.5-fold with SHMT2 silencing in the BE(2)-C cell line compared to control. SHMT2 overexpression decreased MYCN mRNA expression by 1.3-fold. (C) SHMT2 silencing decreased protein expression of phosphorylated Akt-2 (pAkt-2) and Akt-2 compared to control in SK-N-AS cells (top panel). SHMT2 silencing in SK-N-AS cells markedly increased c-Myc protein expression compared to control, while SHMT2 overexpression minimally increased c-Myc protein expression. Densitometry analysis confirmed SHMT2 silencing decreased pAkt-2 by 1.3-fold, Akt-2 protein expression by 2-fold, and c-Myc protein expression by 2.4-fold compared to control (bottom panel). (D) In the BE(2)-C cell lines, N-Myc protein expression was decreased with SHMT2 silencing and overexpression (top panel). SHMT2 silencing did not impact Akt-2 protein expression. SHMT2 overexpression increased pAkt-2 expression, but had no effect on Akt-2 expression. Densitometry analysis, reported as a ratio of each protein band density relative to the density of each ß-actin control band (protein density: ß-actin density), confirmed immunoblotting findings in BE(2)-C cells (bottom panel). SHMT2 silencing resulted in pAkt-2 protein expression 1.6 times lower than control. SHMT2 overexpression increased pAkt-2 protein expression by 1.3-fold. SHMT2 silencing and overexpression had no effect on Akt-2 protein expression. SHMT2 silencing decreased N-Myc protein expression by 1.3-fold and SHMT2 overexpression decreased N-Myc protein expression by 1.8-fold.