Rapamycin and Torin1 effects on neurite outgrowth in presence of HMB.

<p>Cells were pre-treated with rapamycin 20nM or Torin1 10 nM and then treated with 25 μM HMB for 48 h. The inhibitor was maintained during the experiment. Results represent means ± SEM (n = 8). * p<0.05 versus control cells. # p<0.05 versus HMB treated cells.</p

HMB activates mTOR and promotes protein synthesis in Neuro2a cells.

<p><b>(A)</b> Neuro2a cells were pre-treated with rapamycin 20nM or Torin1 10 nM and then were treated with 25 μM HMB for 30 min. Western blot analysis was performed using specific antibodies against phospho- and total-antibodies mTOR. <b>(B)</b> Protein synthesis was measured in Neuro2a cells incubated with 25 μM HMB for 2 hours in the absence (n = 10) or presence of inhibitors of PI3K/Akt (LY294002 20μM), ERK1/2 (PD98059 10μM) or mTOR (rapamycin 20nM). Pretreatment of inhibitors occurred as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135614#pone.0135614.g002" target="_blank">Fig 2</a>. Results represent means ± SEM (n = 4). * p<0.05 versus control cells. # p<0.05 versus HMB treated cells.</p

HMB induces neurite outgrowth in Neuro2a cells.

<p><b>(A)</b> Neuro2A cells were incubated with 25 μM HMB during 72 h and cell viability was measured at different periods of time. <b>(B)</b> Neurite outgrowth was analyzed after 48 h incubation with HMB. Cell morphology was observed under a light microscope (200x) and the neurite bearing cells were counted. Results are plotted as percentage of neurite bearing cells. <b>(C)</b> Acetylcholinesterase activity was measured after 48 h incubation with HMB. <b>(D)</b> Acetylcholinesterase amount was measured by western blot using specific antibodies against AChE. after 48 h incubation with HMB. Results were normalized using actin as loading control. Results are expressed as means ± SEM (n = 4). * p<0.05 versus control cells.</p

HMB induced neurite outgrowth is mediated by PI3K/Akt and ERK1/2 signaling pathways in Neuro2a cells.

<p>Neuro2a cells were treated with 25 μM HMB for 30 min. Western blot analysis was performed using specific antibodies against phospho- and total-antibodies against Akt <b>(A)</b> and ERK1/2 <b>(B)</b>. <b>(C)</b> Neuro2a cells were pre-treated with LY294002 20μM or PD98059 10μM and then treated with 25 μM HMB for 48 h. Inhibitors were maintained during the experiment. Cell morphology was measured as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135614#pone.0135614.g001" target="_blank">Fig 1</a>. Results represent means ± SEM (n = 4). * p<0.05 versus control cells. # p<0.05 versus HMB treated cells.</p

Binding site of 18 (central panel and in green in the others) and comparison with the binding mode of estradiol (Left panel, blue) and ligand AIJ (PDB accession code 1xp9) derived from the dihydrobenzoxathiin scaffold (Right panel, cyan)

<p>Binding site of 18 (central panel and in green in the others) and comparison with the binding mode of estradiol (Left panel, blue) and ligand AIJ (PDB accession code 1xp9) derived from the dihydrobenzoxathiin scaffold (Right panel, cyan)</p

Structures of tested compounds, all compounds possess <i>trans</i> conformation except compounds 19 and 20.

<p>Structures of tested compounds, all compounds possess <i>trans</i> conformation except compounds 19 and 20.</p

Effect estrogen active compounds 15 and 18 and estrogen inactive compounds 11 and 12 on mitochondrial activity (MTT assay), cell mass (SRB assay) and LDH release in estrogen dependent MCF-7 and estrogen independent MDA-MB-231 cells.

<p>Cells were exposed to increasing concentrations of tested compounds for 72 h before performing the assay. Data are reported as % of control and are the means±SEM.</p

Real-Time Phosphate Sensing in Living Cells using Fluorescence Lifetime Imaging Microscopy (FLIM)

Phosphate ions play important roles in signal transduction and energy storage in biological systems. However, robust chemical sensors capable of real-time quantification of phosphate anions in live cells have not been developed. The fluorescein derivative dye 9-[1-(2-methyl-4-methoxyphenyl)]-6-hydroxy-3H-xanthen-3-one (2-Me-4-OMe TG) exhibits the characteristic excited-state proton-transfer (ESPT) reaction of xanthenic derivatives at approximately physiological pH resulting in the dependence of the dye’s nanosecond fluorescence decay time on the phosphate buffer concentration. This allows the 2-Me-4-OMe TG dye to be used with fluorescence lifetime imaging microscopy (FLIM) as a real-time phosphate intracellular sensor in cultured cells. This methodology has allowed the time course of cellular differentiation of MC3T3-E1 murine preosteoblast cells to be measured on the basis of the decrease in the decay time of 2-Me-4-OMe TG. These changes were consistent with increased alkaline phosphatase activity in the extracellular medium as a marker of the differentiation process

Induction of estrogenic activity of HEK293 cells transfected with ER-α receptor by compounds with selective (A) and no selective (B) toxicity against MCF-7 cells. (C) Estrogenic activity of defined compounds toward MCF-7 cell line.

<p>* <i>p</i> < 0.05 compared to the control cells treated with β-estradiol.</p

Estrogenic and cytotoxic activity of tested compounds in HEK293 cells transfected with wild type ERα receptor or plasmids bearing mutated versions of ERα.

<p><b>A</b>.- HEK293 were transfected with wild type ERα receptor or Erα W383A and W383S plasmids and an ER dependent luciferase reporter gene. Results are expressed as mean ± SEM (n = 8). * <i>p</i> < 0.05 was considered statistically significant. <b>B</b>.- HEK293 were transfected mock-transfected or transfected with wild type ERα or Erα W383S plasmids. 48 h after transfection, cells were incubated in the presence of increasing concentrations of compounds 18 for 72 h and cell viability was measured by MTT assay. Results are reported as % viability based on the untreated control cells normalized to 100% viable. Results represent means ± SEM (n = 8). * <i>p</i> <0.05 versus mock-transfected untransfected cells. # p<0.05 versus ERα W383S transfected cells.</p