Research Paper Volume 10, Issue 3 pp 434—462

Induction, regulation and roles of neural adhesion molecule L1CAM in cellular senescence

Figure 5. Interaction of L1CAM with the Erk signaling pathway. (A) Erk 1/2 activity detected as phosphorylation of Erk1/2 (pErk1/2) compared in BJ and A375 cells sorted for L1CAM high and low cell surface level. (B) The effect of L1CAM downregulation using RNA interference on Erk1/2 activity detected by immunoblotting in BJ fibroblasts. (C) L1CAM mRNA level estimated by real time RT-PCR after inhibition of MEK by selumetinib (10 μM; MEKi) in A375 cells. Total (D) and surface L1CAM levels (E) detected by immunoblotting and live cell staining, respectively, in A375 cells after MEK inhibition using selumetinib (10 μM; MEKi). L1CAM mRNA (F) and surface protein level (G) in A375 after downregulation of Erk1/2 using RNA interference (siErk1/2). L1CAM mRNA (H) and total protein (I) levels in control (-DOX) and H-RAS-induced (+DOX) BJ cells before (ctrl) and after inhibition of MEK using selumetinib (10 μM; MEKi). (J) The effect of MEK inhibition by selumetinib (10 μM; MEKi) on the L1CAM total protein level in H-RAS-induced senescent BJ cells sorted for low L1CAM level. Non-template siRNA was used as a control (siNT). For immunoblotting, TFIIH or β-actin were used as a control of equal protein loading. For real time RT-PCR, GAPDH was used as the reference gene. Scale bar, 100 μm. All experiments were performed in three independent replicates. p ˂ 0.05 (*), two-tailed Student’s t-test.