Dephosphorylation of AMPK by PP2A phosphatase promotes neuritogenic effect of SIM in SH-SY5Y cells.

<p>Immunoblot in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074547#pone-0074547-g003" target="_blank">Figure <b>3 (A, B, C and D)</b></a> showing modulation of protein expression of AMPK, p-AMPK (Thr-172), Akt, pAkt (ser-473), pAkt (Thr-308), PP2Ac and PP2Ac (p-Tyr) after SIM treatment for 12 hrs. <b>A</b>) protein expression of AMPK and p-AMPK (Thr-172) at 2 hrs, 6 hrs and 12 hrs after SIM treatment. At 2 hrs a sudden increase in phosphorylation of AMPK was noted which was accompanied by rapid reversal to control. <b>B</b>) AMPK and p-AMPK (Thr-172) levels at 6 hrs in presence of SIM and PI3K inhibitors like wortmannin and LY294002, 10 µM each. No change in pAMPK (Thr-172) was observed among the samples. <b>C</b>) Akt, pAkt (ser-473) and pAkt (Thr-308) levels at 2 hrs, 6 hrs and 12 hrs after SIM addition. A significant (<i>p</i> < 0.05^*) 2.0 fold increase in pAkt (Thr-308) was observed whereas pAkt (Ser-473) displayed no change. <b>D</b>) Time dependent decrease in tyrosine phosphorylation of PP2Ac after SIM treatment till 12 hrs. Total PP2Ac protein levels remained same during SIM treatment. Simultaneously, inhibition of PP2A activity by Fostriecin showed increase in pAMPK (Thr-172) levels in presence or absence of SIM at 6 hrs <b>E</b>) Morphology of SH-SY5Y cells at 10 X magnification under light microscope showing significant (<i>p</i> < 0.001) decline in neuritogenic effect of SIM in presence of Fostriecin and an AMP analogue i.e. AICAR. Error bar graph shows the difference in neurite length between SIM, SIM + Fostriecin and SIM + AICAR treated cells for 12 hrs (mean ± SEM; N = 50 cells per condition from 3 separate cultures). Scale Bar = 100 µm.