Analysis of the clustering quality for the time course data.

<p>A) Outcome of the clustering algorithm, with progressive increase in the number of clusters k: the picture represents, at each different k, the grouping of the 4 couples of alternative primers pointing to the same gene. For k = 2,4,6,8 the alternative primers were correctly grouped together. The “replicas p-value”, on the right, indicates the statistical consistency of the alternative primer grouping, which reaches it maximum value when the algorithm is forced to split the 33 genes into 8 different clusters. On the left, the Z-value of the global clustering, indicating the consistency of the temporal dynamics discrimination. B) Outcome of the algorithm aimed at determining the optimal value for k. The number of clusters N is plotted against a function Θ(N): the minimum of Θ(N), i.e. N = 3, coincides with the optimal value for k. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068078#s4" target="_blank">Materials and Methods</a> section for further details. C) Visual representation, with k = 3, of the distances between trajectories and cluster centroids for all the 33 genes. For each cluster, the genes are disposed at increasing distances from the centroid, proportionally to their normalized Euclidean distances. The distance of the farthest gene is indicated in the proximity of the outer circle. The orientation of the genes reflects the proximity to the remaining two clusters. The distances between the cluster centroids are also indicated.</p

Video_3_Cell Mechanotransduction With Piconewton Forces Applied by Optical Tweezers.AVI

<p>Mechanical stresses are always present in the cellular environment and mechanotransduction occurs in all cells. Although many experimental approaches have been developed to investigate mechanotransduction, the physical properties of the mechanical stimulus have yet to be accurately characterized. Here, we propose a mechanical stimulation method employing an oscillatory optical trap to apply piconewton forces perpendicularly to the cell membrane, for short instants. We show that this stimulation produces membrane indentation and induces cellular calcium transients in mouse neuroblastoma NG108-15 cells dependent of the stimulus strength and the number of force pulses.</p

Graphs of time-course data and their associated functions.

<p>The bold line, representing the average temporal pattern of each cluster, is superimposed to the patterns of the single genes. The histograms depicts the amounts of positive (white bars) and negative (black bars) regulators of the indicated processes for each cluster: n.g., regulation of hippocampal neurogenesis, growth, regulation of growth, surv., regulation of survival, S.T., regulation of synaptic transmission.</p

Video_1_Cell Mechanotransduction With Piconewton Forces Applied by Optical Tweezers.AVI

<p>Mechanical stresses are always present in the cellular environment and mechanotransduction occurs in all cells. Although many experimental approaches have been developed to investigate mechanotransduction, the physical properties of the mechanical stimulus have yet to be accurately characterized. Here, we propose a mechanical stimulation method employing an oscillatory optical trap to apply piconewton forces perpendicularly to the cell membrane, for short instants. We show that this stimulation produces membrane indentation and induces cellular calcium transients in mouse neuroblastoma NG108-15 cells dependent of the stimulus strength and the number of force pulses.</p

Analysis of SRF binding sites by chromatin immunoprecipitation.

<p>Chromatin fragments of hippocampal organotypic cultures were immunoprecipitated with anti-SRF antibody. A) Immunoprecipitation levels normalized to input control: the s.e.m. is calculated over three different replicas. B) Immunoprecipitation of each promoter region, together with input control and IgG antibody, was amplified by PCR. Each sample is derived from three independent replicas.</p

Video_4_Cell Mechanotransduction With Piconewton Forces Applied by Optical Tweezers.AVI

<p>Mechanical stresses are always present in the cellular environment and mechanotransduction occurs in all cells. Although many experimental approaches have been developed to investigate mechanotransduction, the physical properties of the mechanical stimulus have yet to be accurately characterized. Here, we propose a mechanical stimulation method employing an oscillatory optical trap to apply piconewton forces perpendicularly to the cell membrane, for short instants. We show that this stimulation produces membrane indentation and induces cellular calcium transients in mouse neuroblastoma NG108-15 cells dependent of the stimulus strength and the number of force pulses.</p

Characterization of lamellipodial protrusion/retraction cycles and of vertical motion.

<p><b>(a-c)</b> From top to bottom: images of the lamellipodium undergoing cyclic waves of protrusion (t₂) and retraction (t₁ and t₃) in control conditions; the white dotted line represents the leading edge of the lamellipodium_. Scale bar, 5 μm. <b>(d)</b> The profile diagram of the positions of the lamellipodium edge during the time course. Increase in the color intensity shows increase in the frequency of the lamellipodia edge to be present at particular location. White lines used to plot the kymographs. <b>(e)</b> Kymograph showing the protrusion/retraction cycles of lamellipodia. White dots show the leading edge of lamellipodia. The characteristic values of period of protrusion/retraction cycles of lamellipodia motion (black dotted line), the retrograde flow rate (black line) and persistence length of lamellipodia (white line) i.e. (T), (dx/dt) and (dl) respectively were calculated along the label lines. <b>(f)</b> Fractional number of pixels in focus at 5μm height above the coverslip. The protrusion/retraction cycles of lamellipodia is also observed in terms of fractional reached height by lamellipodia. The black line shows the peak position of the fractional height where lamellipodia reach the maximum in axial direction at the end of the retraction.</p

Cellular localization of the functions attributable to the genes up-regulated by GABAr blockage.

<p>Gene functions, extracted from findings reported in the literature, are categorized in four main groups, indicated by the terms: Syn.Tra, regulation of synaptic transmission, growth, regulation of growth, neurogen., regulation of hippocampal neurogenesis, survival, regulation of survival. Each gene is counted as PRO when it positively regulates these processes, while it is counted as ANTI when it negatively affects these processes. The cellular compartment of action for each protein(gene) is chosen according to the indications reported in the literature. For example, it has been shown that the pentraxin Nptx2 (neuronal activity–regulated pentraxin) is localized in the excitatory synapses, where it exerts a homeostatic effect by recruiting AMPAr, AMPA receptors, at excitatory projections onto gabaergic interneurons <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068078#pone.0068078-OBrien1" target="_blank">[118]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068078#pone.0068078-Chang1" target="_blank">[121]</a>.</p

Table containing the list of 33 genes whose up-regulation was confirmed by RT-PCR.

<p>The first column contains the official gene symbol, the second column assigns the role of EF, effector, or TF, transcription factor, while the last column summarizes the validated gene functions, which always refer to tissues or experimental conditions coherent with the present work. For the complete details please refer to the survey presented in the supplementary file S2.</p

Bdnf time course: Graphs representing the three independent replicas of the Bdnf mRna time course.

<p>Time (minutes) on the x-axis, ratio Gabazine/Control on the y-axis. The error-bar plot refers directly to the RT-PCR data. In each of the sub-pictures, it is presented the trend resulting from the single biological replica of the RT-PCR time-course, which is superimposed to the averaged one, calculated as the mean of the three replicates. Thanks to this representation, it is possible to directly compare the single measures (biological replicates) to the averaged one, which eventually was the one used for all of the subsequent clustering analysis. The neurotrophin <i>Bdnf</i>, one of the master regulators of learning and memory, will prove in the end to be up-regulated according to a pattern which is representative for almost 50% of the genes of the set under study.</p