Influence of CdCl₂ (1 µM), an unspecific blocker of HVA and LVA VGCC on growth cone collapse rate and changes in Sema3A (100 ng/mL) induced intracellular calcium concentration.

<p>A) Growth cone collapse rate in control group (black bar) Sema3A treated group (red bar), CdCl₂ treated group (blue bar) or both treatments (green bar). B) Changes of relative Fura-2 340/380 fluorescence in control conditions (black curve) and 1 µM of CdCl₂ (blue curve) followed by DRG neuron treatment with Sema3A (red and green curves respectively) at indicated time point. All data have been normalized to the point, before Sema3A was added (this point equals to 1 arbitrary unit AU). C) Bars represent mean ± SEM of relative Fura2 fluorescence: black bar represent control Fura-2 fluorescence; red bar represent mean fluorescence in presence of Sema3A; blue bar indicates condition in presence of CdCl₂ and green bar indicates conditions where Sema3A was added in presence of CdCl₂; ***p<0.001; **p<0.01. In the panels A and C upper, middle and lower lines represent statistical difference of the corresponding groups from control, from Sema3A, and CdCl₂ groups respectively.</p

Growth cone morphology in control conditions and after application of Sema3A.

<p>Same region of time-laps microscopy represents typical morphology change of growth cones grown for 23 hours in control conditions and after 60 minutes incubation with 100 ng/mL of Sema3A.</p

Data from gene expression profiling of E15 mouse DRG’s in two different conditions: control conditions (Control), and bath application of 100 ng/mL Sema3A (Sema3A).

<p>Signal strength columns of Control and Sema3A conditions represent average values of three independent experiments. Fold change – fold change of mRNA expression in different conditions, p value – Student’s t-test for significance of difference.</p

Change of Sema3A (100 ng/mL) induced Fura-2 fluorescence 340/380 ratio, corresponding to change of [Ca²⁺]_i concentration in different parts of sensory neurons.

<p>Effect of addition of Sema3A concentration was measured recording the same ROIs corresponding to either axon parts proximal to growth cones (panels A and B) or neuron soma (panels C and D). All data have been normalized to the fluorescent ratio at the time point preceding Sema3A addition (the value of 340/380 ratio at this point equals to 1 arbitrary unit AU). A) Curve indicates the relative fluorescence of ROIs of axons (black curve) and after (red curve) the addition of Sema3A. B) Bars represent the mean ± SEM of 340/380 ratio in axons in absence (black bar) and presence (red bar) of Sema3A. C) Curve indicates the relative fluorescence of ROIs of neuron soma before (black curve) and after (red curve) the addition of 100 ng/mL of Sema3A. D) Bars represent the mean ± SEM of 340/380 ratio in neuron soma in (black bar) and presence (red bar) of Sema3A. To reveal statistical significance Dunn’s post hoc test for Kruskal-Wallis analysis was performed. In the panels B and D lines represent statistical difference of control and Sema3A of the same ROIs evaluated. Here “ns” denotes not significant.</p

Influence of nifedipine (10 µM), a selective blocker of HVA L-type VGCC on growth cone collapse rate and changes in Sema3A (100 ng/mL) induced intracellular calcium concentration.

<p>A) Growth cone collapse rate in control group (black bar) Sema3A treated group (red bar), nifedipine treated group (blue bar) or both treatments (green bar). B) Changes of relative Fura-2 340/380 fluorescence in control conditions (black curve) and 10 µM of nifedipine (blue curve) followed by DRG neuron treatment with Sema3A (red and green curves respectively) at indicated time point. All data have been normalized to the point, before Sema3A was added (this point equals to 1 arbitrary unit AU). C) Bars represent mean ± SEM of relative Fura2 fluorescence: black bar represents control Fura-2 fluorescence; red bar represents mean fluorescence in presence of Sema3A; blue bar indicates condition in presence of nifedipine and green bar indicates conditions where Sema3A was added in presence of nifedipine. Here S3A and Nif corresponds to Sema3A and nifedipine respectively; ***p<0.001; ns–not significant. In the panels A and C upper, middle and lower lines represent statistical difference of the corresponding groups from control, from Sema3A, and nifedipine groups respectively.</p

Increase of Sema3A induced Fura-2 fluorescence 340/380 ratio, corresponding to increased [Ca²⁺]_i concentration in growth cones.

<p>Two different protocols are shown. In panel A, different dishes were used to evaluate the Sema3A concentration dependent effect, while in panel C, the effect of addition of supplemental 100/mL Sema3A concentration was measured recording the same ROIs. All data have been normalized to the fluorescent ratio at the time point preceding Sema3A addition (the value of 340/380 ratio at this point equals to 1 arbitrary unit AU). A) black curve indicates the relative fluorescence before the addition of Sema3A at indicated concentrations (light red curve and dark red curve). B) Bars represent the mean ± SEM of 340/380 ratio in presence of different Sema3A concentrations. C) Curve indicates relative fluorescence in control condition before addition of 100 ng/mL Sema3A following by addition of 100 ng/mL Sema3A (final concentration 200 ng/mL) at indicated time points. D) Bars represent the mean ± SEM of 340/380 ratio in presence of different Sema3A concentrations. To reveal statistical significance Dunn’s post hoc test for Kruskal-Wallis analysis was performed. In the panels B and D upper and lower lines represent statistical difference of the corresponding groups from control and from 100 ng/mL Sema3A respectively. Here *** denotes p<0.001.</p

Influence of NiCl₂ (100 µM), a blocker of T and R type LVA VGCC on growth cone collapse rate and changes in Sema3A (100 ng/mL) induced intracellular calcium concentration.

<p>A) Growth cone collapse rate in control group (black bar) Sema3A treated group (red bar), NiCl₂ treated group (blue bar) or both treatments (green bar). B) Changes of relative Fura-2 340/380 fluorescence in control conditions (black curve) and 100 µM of NiCl₂ (blue curve) followed by DRG neuron treatment with Sema3A (red and green curves respectively) at indicated time point. All data have been normalized to the point, before Sema3A was added (this point equals to 1 arbitrary unit AU). C) Bars represent mean ± SEM of relative Fura2 fluorescence: black bar represent control Fura-2 fluorescence; red bar represent mean fluorescence in presence of Sema3A; blue bar indicates condition in presence of NiCl₂ and green bar indicates conditions where Sema3A was added in presence of NiCl₂; ***p<0.001; ns–not significant. In the panels A and C upper, middle and lower lines represent statistical difference of the corresponding groups from control, from Sema3A, and NiCl₂ groups respectively.</p

Binding properties as well as cellular and pore-forming activities of wild-type and LukS-PV mutants¹.

1<p><i>K_i</i> values, calcium entry slope and ethidium entry at 30 min were obtained for wild-type LukS-PV and all mutants with hPMNs. <i>K_i</i> values with U937-C5aR cells were obtained for wild-type LukS-PV and for the most affected mutants (values given in parenthesis).</p>2<p>Mutations causing a significant decrease in LukS-PV affinity for hPMNs (<i>p</i><0.001, one-way ANOVA with Dunnett's post test).</p

Molecular surface of the rim domain of LukS-PV.

<p>Residues identified in this study as important for the binding of LukS-PV on the C5a receptor (<i>K_i</i> increased more than 50 fold upon mutation to Ala) are depicted in red. Mutated residues affecting binding to a lesser extent (i.e. increase in <i>K_i</i> by a factor between 5 and 50) are depicted in pink whereas residues for which no effect on binding was found upon mutation (increase in <i>K_i</i> less than 3 fold) are depicted in blue (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092094#pone-0092094-t001" target="_blank">Table 1</a>). Two orthogonal views around a vertical axis are presented, with the orientation on the left being the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092094#pone-0092094-g005" target="_blank">figure 5</a>.</p

Binding properties of LukS-PV* and LukS-PV mutants to hPMNs and U937-C5aR cells.

<p>A. Flow cytometry measurement of LukS-PV* and fluorescein-labeled LukS-PV G10C binding to human PMNs and U937-C5aR cells (<i>n</i> = 3). B. Graphic representation of the <i>K</i>_i values obtained for wild-type or mutant LukS-PV. The dotted line corresponds to the value of wild-type LukS-PV. Error bars represent the 95% confidence interval. Statistical analysis: **: <i>p</i><0.01, ***: <i>p</i><0.001 (<i>n</i> = 3).</p