Phosphoproteome Profiling of SH-SY5y Neuroblastoma Cells Treated with Anesthetics: Sevoflurane and Isoflurane Affect the Phosphorylation of Proteins Involved in Cytoskeletal Regulation - Table 1

<p>Phosphoproteome Profiling of SH-SY5y Neuroblastoma Cells Treated with Anesthetics: Sevoflurane and Isoflurane Affect the Phosphorylation of Proteins Involved in Cytoskeletal Regulation</p> - Table

Reorganization of the microfilament cytoskeleton in anesthetic-treated SH-SY5y cells.

<p>Cells were cultured on glass coverslips and incubated for the indicated times with vehicle or 1 mM sevoflurane and isoflurane. The redistribution of filamentous actin and globular actin was determined by phalloidin–halloidinne aDNAse I-Alexa488 staining and immunofluorescence microscopy. A. Magnification, 400 × (the scale bar represents 50 μm); B. Magnification, 1,000 × (the scale bar represents 20 μm). Similar results were obtained in three independent experiments.</p

Typical kinetics of protein phosphorylation regulated in response to sevoflurane treatment in SH-SY5y cells.

<p>The kinetics of phosphorylation changes over time is depicted for selected proteins (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162214#pone.0162214.t001" target="_blank">Table 1</a>). Each value represents single values from each gel. Lines connect average standardized abundance values. The Y axis represents the log ratio of treatment per control.</p

SH-SY5y cell phosphoprotein patterns following treatment with sevoflurane monitored by 2D gel electrophoresis.

<p>SH-SY5y cells were incubated with sevoflurane for 2, 5, 15, or 30 min; harvested and lysed. Intracellular enriched phosphoproteins were analyzed by 2D gel electrophoresis. Right panel: Coomassie Brilliant Blue staining; left panel: ProQ Diamond. Y axes represent the apparent molecular mass (kDa), and X axes represent pH values. Acquired images showed reproducibility of experiments. Data shown are representative of three separate experiments.</p

PAK-dependent rearrangement of actin filaments induced by sevoflurane and isoflurane.

<p>(A) Cells were cultured on glass coverslips and pretreated with IPA3 (5 μM) or FRAX597 (50 nM) for 30 min and subsequently exposed to 1 mM sevoflurane or isoflurane for 30 min. The redistribution of filamentous actin and globular actin was determined by phalloidin–Alexa568 and DNAse I-Alexa488 staining and immunofluorescence microscopy. Magnification, 400 × (the scale bar represents 50 μm). Similar results were obtained in three independent experiments. (B) Quantification of fluorescence intensity was performed in 25 randomized districts from each treatment group. Quantification was performed for four independent experiments. Results were expressed as F-actin activity relative to that of G-actin and statistical analysis was performed. Results are shown as means ± SD of 100 randomized districts obtained from four independent experiments. Statistical significance was determined using the Student’s t-test compared with the untreated control. Asterisks indicate statistically significant differences between treatment and control (*<i>p</i> < 0.01).</p

Quantification of fluorescence intensity of F-actin and G-actin.

<p>Quantification of fluorescence intensity was performed from 25 randomized districts on culture plates from each treatment group (control, 5, 15, and 30 min). Quantification was performed for four independent experiments. Results were expressed as F-actin activities relative to the one of G-actin and statistical analysis was performed. Results are shown as means ± SD of 100 randomized districts obtained from four independent experiments. Statistical significance was determined using the Student’s <i>t</i>-test compared with the control. Asterisks indicate statistically significant difference between 5, 15, 30 min treatments and control (*<i>p</i> < 0.01, **<i>p</i> < 0.05).</p

Phosphoproteome Profiling of SH-SY5y Neuroblastoma Cells Treated with Anesthetics: Sevoflurane and Isoflurane Affect the Phosphorylation of Proteins Involved in Cytoskeletal Regulation

<div><p>Inhalation anesthetics are used to decrease the spinal cord transmission of painful stimuli. However, the molecular or biochemical processes within cells that regulate anesthetic-induced responses at the cellular level are largely unknown. Here, we report the phosphoproteome profile of SH-SY5y human neuroblastoma cells treated with sevoflurane, a clinically used anesthetic. Phosphoproteins were isolated from cell lysates and analyzed using two-dimensional gel electrophoresis. The phosphorylation of putative anesthetic-responsive marker proteins was validated using western blot analysis in cells treated with both sevoflurane and isoflurane. A total of 25 phosphoproteins were identified as differentially phosphorylated proteins. These included key regulators that signal cytoskeletal remodeling steps in pathways related to vesicle trafficking, axonal growth, and cell migration. These proteins included the Rho GTPase, Ras-GAP SH3 binding protein, Rho GTPase activating protein, actin-related protein, and actin. Sevoflurane and isoflurane also resulted in the dissolution of F-actin fibers in SH-SY5y cells. Our results show that anesthetics affect the phosphorylation of proteins involved in cytoskeletal remodeling pathways.</p></div

Phosphorylation levels of immunoprecipitated HSP90β, nucleolin, Ras-GAP SH3 binding protein, and matrin 3 in SH-SY5y cells exposed to sevoflurane and isoflurane.

<p>After cell lysates were incubated with each primary antibody overnight at 4°C, Protein A/G Plus was added and complexes incubated for 4 h at 4°C. Pellets were washed, and immunoprecipitated complexes were released and analyzed by western blotting using phosphospecific antibodies. Semi-quantitative analysis was performed using densitometry, and results are expressed as activity relative to that of the control. Results are shown as means ± SD of three independent experiments. Statistical significance was determined using the Student’s <i>t</i>-test compared with the control. Asterisks indicate statistically significant difference between 5, 15, 30 min treatments and the control (*<i>p</i> < 0.01). For nucleolin, statistical significance was additionally determined using one-way ANOVA compared with the 5 min activated level. Sharps indicate the statistically significant difference between 5 min treatment and after 15 min (#<i>p</i> < 0.01).</p