MOESM1 of Neuroprotection by chitosan nanoparticles in oxidative stress-mediated injury

Additional file 1. Chitosan nanoparticles. Transmission electron micrographs of chitosan nanoparticles. Most particles were 100 nm or less in diameter. Corresponding table shows Chi-DSNPs did not significantly inhibit cell proliferation after 20 h incubation at different concentrations (0, 0.1, 0.2, 0.5 mg/ml)

Expression patterns of innexins functioning in <i>C. elegans</i> body-wall muscle.

<p>Expression patterns were assessed by analyzing GFP signal in live worms expressing innexin promoter and GFP transcriptional fusions. Body-wall muscle expression (indicated by arrows) was observed for four of the six innexins important to muscle electrical coupling, including <i>unc-9, inx-10, inx-11</i> and <i>inx-18</i>. The <i>unc-9</i> expression data were shown previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076877#B33" target="_blank">33</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076877#B34" target="_blank">34</a>].</p

Speculative models of muscle gap junctions.

<p>These highly speculative models are used to illustrate three possible stoichiometries out of the myriad possibilities. See the text for reasons for speculating these models.</p

Junctional conductance (<i>G</i>_<i>j</i>) of <i>C. elegans</i> body-wall muscle was significantly decreased in mutants of six innexins.

<p><b>A</b>. Diagram showing ventral body-wall muscle cells. Ventral muscles include the left and right quadrants with each quadrant consisting of two rows of muscle cells in a monolayer. The highlighted cell pairs (L1/L2 and R1/R2) represent those used for electrophysiological analyses. <b>B</b>. <i>G</i>_<i>j</i> was indistinguishable between wild type (WT) and mutants of 16 innexins. <b>C</b>. Mutants of 6 innexins showed significantly lower <i>G</i>_<i>j</i> when compared with WT, and the coupling defect was rescued completely by expressing a wild-type innexin in each corresponding mutant. The asterisk (*) indicates a statistically significant difference compared with WT. The number on each bar represents the number (<i>N</i>) of cell pairs analyzed.</p

Stomatin and Panx1 were both enriched in the plasma membrane region.

<p>Myc-tagged Panx1 (Panx1::Myc) and HA-tagged stomatin (Stomatin::HA) were coexpressed in HEK-293 cells. Panx1 and stomatin were visualized by double immunostaining with antibodies to Myc and HA, respectively. Shown are representative images of Panx1::Myc (green), Stomatin::HA (red), and the merged picture. A selected region in the top panel was magnified and shown in the bottom panel.</p

Stomatin inhibited Panx1 channel-mediated outward whole-cell currents in transfected HEK-293 cells.

<p>HEK-293 cells were transfected independently with Panx1, stomatin, Panx1 plus stomatin, and empty vectors (Control). Whole-cell currents in response to a voltage ramp (–60 to +90 mV over 10 sec) were recorded from the transfected cells either in the absence or presence of the hemichannels blockers carbenoxolone (CBX 25 µM) or ¹⁰Panx1 (100 µM). <b>A.</b> Averaged current traces in response to a voltage ramp (–60 to +90 mV over 10 sec). Cells expressing Panx1 showed large voltage-dependent outward currents compared with the Control, which were blocked by either CBX or ¹⁰Panx1 and reduced by stomatin. Note that the Control current trace is almost invisible due to overlap with other traces. <b>B.</b> Statistical comparisons of the whole-cell current density at indicated membrane voltages. The asterisk (*) indicates a significant difference compared with the Control whereas the pound sign (#) indicates a significant difference compared with the Panx1 group (<i>p</i><0.01, one-way ANOVA with Bonferroni posthoc tests).</p

Stomatin did not alter total or cell surface expression of Panx1.

<p>The amount of total and surface Panx1 protein levels were determined with transfected HEK-293 cells expressing either Myc-tagged Panx1 (Panx1::Myc) alone or Panx1::Myc plus HA-tagged stomatin (Stomatin::HA). The bar graphs show the densities of the Panx1::Myc total and surface protein bands (normalized by actin) based on 5 independent experiments.</p

Inhibition of stomatin expression enhanced Panx1-dependent outward whole-cell currents in astrocytes.

<p>Cells were transfected with either stomatin siRNA or scrambled siRNA. <b>A.</b> Effects of scrambled and stomatin siRNA on stomatin mRNA level as determined by real-time PCR (from 3 independent experiments). The asterisk (*) indicates a significant difference compared with scrambled siRNA (<i>p</i><0.01, paired <i>t</i>-test). <b>B.</b> Averaged current traces in response to a voltage ramp (–60 to +90 mV over 10 sec) showing the effects of siRNA and the specific Panx1 channel inhibitor ¹⁰Panx1. <b>C & D.</b> Comparisons of the peak outward (at +90 mV) and inward (at –60 mV) currents among the different groups. The asterisk (*) indicates a significant difference (<i>p</i><0.01) compared with scrambled siRNA whereas the pound sign (#) indicates a significant difference (<i>p</i><0.05) compared with stomatin siRNA (one-way ANOVA with Bonferroni posthoc test).</p

Stomatin did not inhibit Panx1-dependent ethidium uptake in transfected HEK-293 cells.

<p>HEK-293 cells were transfected with Panx1, stomatin, Panx1 plus stomatin, or empty vectors (Control). <b>A.</b> Transfected cells were incubated with either phosphate-buffered saline (1 mM K⁺) or 150-mM [K⁺] solution containing ethidium bromide (20 µM) for 5 min. Fluorescence intensity of the cells (identified by GFP fluorescence) at 5 min of the incubation was background subtracted and compared among the different groups. The asterisk (*) indicates significant difference compared with the Control while the pound sign (#) indicate significant difference compared with the Panx1 group at 1 mM [K⁺]_o (<i>p</i><0.01, one-way ANOVA with Bonferroni posthoc tests). The numbers inside the columns indicate the number of cells analyzed. <b>B.</b> Transfected cells were voltage-clamped to +80 mV (5 sec) and –60 mV (1.5 sec) alternatively for a total duration of 2 min immediately following the addition of ethidium (20 µM) to the bath solution. The brief sojourns to –60 mV were necessary for maintaining the whole-cell configuration. Fluorescence intensities of the cells at the beginning and end of the voltage pulses were imaged. The red horizontal lines in the graph indicate the means. Fluorescence intensities are shown in arbitrary units in the plots of both A and B.</p