Fig. 4

AChE inhibition suppresses ERK & p38 phosphorylation and inhibits cell cycle progression. a Bar chart depiction of differences in the signal intensity of various kinases from the human phosphokinase screen in T3M4 cells treated with physostigmine (physo) vs untreated controls. b-c Representative dot blots and selected bar graphs from the human phosphokinase screen in T3M4 cells treated with physostigmine (physo) vs untreated controls. d Western Blot analysis of phosphorylated ERK (pERK)/ERK in T3M-4 cells after 5 min-treatment with phorbol 12-myristate 13-acetate (PMA, an ERK-activator/positive control, Sigma-Aldrich, Taufkirchen, Germany), U0126 (a MEK-inhibitor/negative control, Sigma-Aldrich, Taufkirchen, Germany), physostigmine/Phys and pyridostigmine/Pyr at low, middle and high concentrations (1 ng/μl, 10 ng/μl and 30 ng/μl, respectively). Graph shows quantification of the densitometry of immunoblot for phosphorylated ERK (pERK)/ERK shown in percent of expression compared to control (Pyr. 5 min [high] **p = 0.004 by unpaired t-test). e-h Western Blot analysis of phosphorylated ERK (pERK)/ERK, of phosphorylated p38 (pp38)/p38, phosphorylated Src (pSrc)/Src and phosphorylated AMPKα (pAMPKα)/AMPKα in SU86.86 cells after 5 min-treatment with phorbol 12-myristate 13-acetate (PMA, an ERK-activator/positive control, Sigma-Aldrich, Taufkirchen, Germany), U0126 (a MEK-inhibitor/negative control, Sigma-Aldrich, Taufkirchen, Germany), physostigmine/Phys and pyridostigmine/Pyr at low, middle and high concentrations (1 ng/μl, 10 ng/μl and 30 ng/μl, respectively). Graphs shows quantification of the densitometry of immunoblots (pERK: Phys. 5 min [low] *p = 0.0109; Phys. 5 min [mid], Phys 5 min [high], Pyr. 5 min [mid] and Pyr. 5 min [high] ****p < 0.0001 by unpaired t-test; pp38: Phys. 5 min [low] **p = 0.0019; Phys. 5 min [mid] *p = 0.0189; Phys. 5 min [high] *p = 0.0302 by unpaired t-test). n.s.: not significant. i Scatter plot of propidium iodide (PI) signal in T3M4 PCC untreated (upper graph) and treated (lower graph) with 30 ng/μl of physostigmine and their corresponding cell count plot. j shows cell count distribution throughout G1/0-, S- and G2/M-phases of T3M-4 cells. G1/0: ACh 1000 μM *p = 0.0122, Pyridostigmine 30 ng/μl **p = 0.0017, Phys. + Pyr. 15 ng/μl + 15 ng/μl **p = 0.0046; S: ACh 1000 μM **p = 0.0033, Physostigmine 30 ng/μl *p = 0.0139 by unpaired t-test). Mean ± SD. *p < 0.05, **p < 0.01, ****p < 0.0001