Transcriptional profiling and Ingenuity Pathway Software analysis
To gain evidence for the hypothesis that SMF exposure activates or otherwise modulates signaling networks, human embryoid body derived (hEBD) cells [24
] were exposed to 0.23–0.28 T fields and mRNA microarray profiling was used to determine changes to global patterns of gene expression. In the first tests, 15 min SMF exposure (followed by one day recovery) was tested based on reports that gene expression responded to magnetic exposure this quickly [25
]. In our evaluation, however, only two genes were up- and down-regulated with a statistical probability > 95% (Table ) and none met the common benchmark of a 2- (or even 1.75-) fold change. Nonetheless, the reproducibility over multiple probes for the same gene indicated that these modest changes were real and provided impetus to investigate longer term exposure.
Microarray profiling of mRNA levels in hEBD cells exposed to SMF for 15 min.
Indeed, after one day (~24 h) of SMF treatment, 379 genes were up-regulated and 549 were down-regulated with statistical significance (Figure ); even greater changes were seen after 4 or 5 days of exposure. The magnitude of the change for most genes, however, was modest (Figure ) with only 7 showing up-regulation ≥ 2-fold (Figure ) and 20 showing a similar degree of down-regulation (Figure ). After 5 days of continuous SMF exposure, the number of genes up-regulated by ≥ 2-fold increased to 85 (Figure ) while 94 were down-regulated to a similar extent. Interestingly, in an experiment where the cells were allowed to recover for one day under normal culture conditions after prolonged SMF exposure, the number of genes that remained up-regulated by ≥ 2-fold fell by almost half (from 85 to 47, Figure ) whereas the number of down-regulated genes increased by 35 (Figure ).
Figure 2 mRNA profiling of SMF-treated hEBD cells. (A) The magnitude and number of genes that showed statistically significant changes in mRNA levels after SMF exposure compared to untreated control cells are given (four data points, representing genes with ≥ (more ...)
The microarray results were consistent with the activation of signal transduction pathways over the short term (i.e., in less than one day) leading to an amplified set of genetic changes over the next several days. A simple inspection of transcriptional changes (for example, the top 5 up- and down-regulated genes under each exposure condition listed in Tables , , and ) did not lead to any obvious insights into the over-riding effects of SMF however. Therefore, to flesh out this hypothesis, the Ingenuity Pathway Analysis software tool [21
] was used to analyze the microarray data resulting in the identification of nine networks that responded to SMF exposure in hEDB LVED cells (Table ; data analysis is shown for cells subject to five days of continuous SMF exposure and the annotated networks are provided in Additional file 1
). Several of these pathways reflected known biological responses to magnetic exposure. For example, changes to intracellular Ca2+
pools observed in cell lines exposed to SMF [18
] were consistent with interleukin-6 (IL-6) centered signaling responses (ID#2, Table ) mediated through the ability of this cytokine to be modulated by Ca2+
]. Similarly, Wnt responses (ID#6, Table ) can be activated by a non-canonical Ca2+
dependent mechanism [29
]. Moving above the cell level, two networks were identified (ID#3 and ID#5) that related to cardiovascular development and hematological function, respectively, and thus dovetail with a recent report by Morris and Skalak where SMF exposure of 0.06–0.14 T for a comparable time period (seven days) facilitated micro-vessel regeneration after surgical intervention [13
]. Likewise, Strieth and coauthors have reported that SMF affects the vascular and blood flow [30
] and Okano and coworkers have investigated the modulation of blood vessels by magnetic fields [10
Gene expression for hEBD LVEC cells exposed to SMF for one day (i.e., "Group 2") compared to control cells incubated without SMF exposure (Group 1).
Gene expression for hEBD LVEC cells exposed to SMF for five days (i.e., Group 3) compared to control cells incubated without SMF exposure (Group 1).
Gene expression for hEBD LVEC cells exposed to SMF for four days followed by one day of recovery (i.e., Group 4) compared to cells incubated without SMF exposure (Group 1).
Signaling networks identified to respond to SMF exposure through data analysis with the Ingenuity Pathway Analysis software tool.1
SMF increased IL-6 mRNA levels and protein secretion at early time points
Even though the software analysis of the microarray data was consistent with a mechanism wherein SMF acted as a stimulus for signaling pathways, limitations of this methodology precluded any firm conclusions. Signaling pathway responses, for example, are typically measured over time intervals of minutes to hours and require evaluation with closely-spaced time points not practical by microarray profiling over several days. Therefore, to verify that the transcriptional changes we observed represented legitimate responses to SMF, we selected IL-6 for conventional biochemical characterization. Of the nine networks identified by microarray profiling, the selection of IL-6 for additional scrutiny was based on several factors. First, a recent report linked 0.4 T SMF exposure to increased IL-6 production in fibroblasts [34
] and plausible membrane-based modes of activation IL-6 (e.g., through Ca2+
or TLR4) exist. Furthermore, reports that SMF can promote differentiation [20
] – coupled with the propensity of the hEBD LVEC line used in this study to display neural markers [24
] together with reports that IL-6 promotes astrocytogenesis [35
] – offered the possibility that cell-level responses (e.g., differentiation of the hEBD cells to astrocytes) could be observed in these experiments.
Biochemical validation began by quantitative real-time polymerase chain reaction (qRT-PCR) analysis of IL-6 mRNA levels over the first 24 h of SMF exposure, a time frame selected based on the numerous changes seen in the microarray data after one day (Figure ) and literature reports of biphasic IL-6 activation during this time period [36
]. In these experiments, IL-6 mRNA levels increased two hours into SMF exposure and remained elevated compared to untreated control cells at 4, 7 and 24 h (Figure ). IL-6 secretion into the culture medium followed slower kinetics, first showing a measurable increase at 7 h after which SMF-treated cells out-produced control cells up to 96 h (Figure ). The SMF-exposed cells experienced the largest relative increase compared to untreated controls at 48 h, followed by a decline to slightly less than control levels at the end of the six day monitoring period.
Figure 3 Response of interleukin-6 (IL-6) to SMF exposure and putative activating mechanisms in hEBD LVEC cells. (A) Levels of IL-6 mRNA were determined by qRT-PCR during the first 24 h of SMF exposure. (B) IL-6 concentrations in the culture medium of SMF-exposed (more ...)
TLR4 was activated by SMF in tandem with IL-6
Upon verifying that IL-6 was activated by SMF at both the mRNA and protein levels, we sought more detailed insight into this response. As indicated in Figure & (for perspective, Figure summarizes the connections between SMF, IL-6 and other pathways elements and cellular outcomes described in this report), IL-6 activation was consistent with the known ability of magnetic fields to alter calcium ion channel flux and reports of Ca2+
-dependent up-regulation of IL-6 (the impact of SMF on calcium flux was experimentally verified for currently-used hEBD LVEC cells, Figure ). In addition, connections IL-6 shares with TLR4 [37
], combined with the dependence of the signaling activity of Toll-like receptors on their lateral diffusion within membrane microdomains [19
], suggested a parallel route through which SMF could influence IL-6. Specifically, a sequence of events can be postulated where SMF changes membrane fluidity thereby modulating TLR4 (Figure ) and downstream IL-6 responses (Figure ) through a Ca2+
-dependent mechanism (Figure ) or through TLR4-mediated p38 phosphorylation (Figure ).
Experimentally, because TLR4 transcription is strictly auto-regulated in a stimulus-dependent manner ([38
] and Figure ), qRT-PCR can be used to monitor its activation. By monitoring this endpoint, we found that TLR4 transcript levels increased during the first several hours of SMF exposure (Figure ). Interestingly, self-activation of TLR4 can lead to either the down-regulation of its mRNA (as seen in rat glial [38
] or murine macrophages [42
]) or to up-regulation (as seen in murine lung [37
] or human monocytes and polymorphonuclear leukocytes [43
]); the current up-regulation of TLR4 mRNA observed in SMF-treated embryonic cells is consistent with results obtained in other types of human cells upon activation of TLRs.
SMF activation of TLR4 impinges upon MAPK pathways
The activation of IL-6 in cells exposed to SMF was consistent with signal transduction through the upstream involvement of TLR4 (Figure &). To gain biochemical evidence for this connection, we analyzed the phosphorylation of p38, which lies in the pathway that connects TLR4 with IL-6, and found the predicted increase in phosphorylated p38 in SMF treated cells (Figure ). This result, in addition to establishing a connection between IL-6 and SMF through TLR4, provided evidence that SMF impinges on MAPK signaling (p38 plays a central role in mediating MAPK responses) prompting us to evaluate changes to proliferation and apoptosis. In these experiments a significant reduction in proliferation was seen for hEBD LVEC cells after three days of SMF exposure; this effect lessened by the sixth day and was lost by the ninth day (Figure ). Qualitatively, this short term change in proliferation was consistent with studies where SMF transiently altered proliferation [44
]. Annexin/propidium iodide staining assays showed that reduced proliferation during early phases of SMF exposure was not a consequence of increased apoptosis (Figure &) in agreement with reports that SMF, if anything, is protective against apoptosis [18
]. Having ruled out that the SMF treated cells were dying, a plausible explanation for the reduced proliferation was that the cells were undergoing differentiation with a concomitant decrease in their growth rate; this possibility was supported by data presented later in this report.
Figure 4 MAPK-related responses to SMF in hEBD LVEC cells. (A) An increase in p38 phosphorylation occurred in the SMF-treated cells (representative results from one of three experiments is shown for 30 min of SMF exposure; a normalized ratio of phosphorylated (more ...)
SMF responses are cell line dependent
As a brief diversion from the main thrust of this study, which was to connect SMF with cellular responses associated with IL-6 in human embryonic cells, we wish to emphasize that the impact of SMF on other common laboratory cells such as the Jurkat, HeLa, and HEK AD293 lines was surveyed and "obvious" effects such as pronounced changes in proliferation (as seen in Figure for the hEBD LVEC line) or altered morphology (as shown later in this report) were not observed. For example, representative data is shown for the HEK AD293 line in Figure where control and SMF-exposed cells had identical growth rates when measured by either the MTT assay (Panel A) or through cell counting (Panel B). The clear-cut differences seen between the embryonic hEBD LVEC line and cancer lines were not surprising based on reports that even closely-matched cell lines respond uniquely to SMF [46
]; instead these findings support the hypothesis that changes to Ca2+
flux (as shown in Figure ) – a parameter that is highly cell line dependent [18
] – contributes to the cellular responses we observed in the cells exposed to SMF.
Figure 5 Proliferation is not altered by SMF in HEK AD293 cells. The cell line dependence of SMF effects is illustrated by the lack of a response in SMF-treated HEK AD293 cells when proliferation was measured by the MTT assay (Panel A) or by cell counts (Panel (more ...)
Connections between gangliosides and IL-6 exist in hEBD LVEC cells
To gain insight into whether regulatory networks beyond TLR4 or calcium flux contributed to the up-regulation of IL-6 in SMF treated cells, we next focused on gangliosides [38
]. Gangliosides are sialic acid-bearing glycosphingolipids (GSLs) that are integral components of lipid rafts and caveolae of the type surrounding TLR4 that not only organize these microdomains but also regulate the signaling functions of embedded proteins (as discussed in more detail in review articles [48
]). Consequently, TLR4 [38
] and IL-6 [47
] can be influenced by the equilibrium between the 'inert' (in this context) GSL lactosylceramide (LacCer) and the suppressive ganglioside GM3 (Figure ).
Before beginning experiments to probe the impact of SMF exposure on gangliosides, the relationship between GM3 (and its disialylated derivative GD3) and IL-6 was first investigated to establish a baseline for the hEBD LVEC line (before the current study, there was negligible literature precedent for a connection between GSL and IL-6 in human embryonic cells). A long-lasting and substantial (e.g., > 95% at 4 d) reduction in IL-6 mRNA was observed in cells incubated with exogenously-added GM3 or GD3 (Figure ). Crosstalk between gangliosides and IL-6 also held in the reverse direction as demonstrated by a dose dependent decrease in GM3 in cells incubated with exogenously-added IL-6 (Figure ). By reducing the amount of GM3 present in a cell (Figure ), IL-6 can alleviate the suppressive effects of this ganglioside on its transcription (Figure ) thus setting up a 'feed-forward' loop that offers an mechanistic explanation for the self-activation of IL-6 described in the literature [50
] and demonstrated for hEBD LVEC cells in this study (Figure ). Figure shows that levels of TLR4 mRNA also increased significantly in IL-6 supplemented cells consistent with the removal of concomitant inhibitory effects of GM3 on TLR4 [38
]. Together with the impact of SMF on IL-6 shown in Figure , these results demonstrate that SMF has the capacity for tuning IL-6 signaling by adjusting the relative proportions of the 'active' ganglioside GM3 and its 'inert' asialo counterpart LacCer (Figure ) thereby contributing to the transcriptional up-regulation of TLR4 and IL-6 (Figure &).
Figure 6 Crosstalk between IL-6 and gangliosides in hEBD LVEC cells. (A) IL-6 mRNA decreased upon supplementation of the culture medium with 5.0 μM of either GM3 or GD3 (n ≥ 3; p < 0.01 for all time points shown compared with controls that (more ...)
IL-6 mediated changes to GM3 and GD3 occur via NEU3 and STGAL5
Mechanistically, changes to one of two enzymes could explain the shift in equilibrium away from the suppressive ganglioside GM3 to its inert asialo counterpart LacCer (Figure ); specifically, an increase in the recycling enzyme NEU3 or a decrease in the biosynthetic enzyme ST3GAL5 (Figure ). Despite no previously-known direct links between IL-6 and ST3GAL5 or NEU3, increased phosphorylation of ERK1/2 has been connected with the up-regulation of STGAL5 ([51
], as shown in Figure ). Therefore, based on a report linking IL-6 and MAPK signaling through JAK/STAT that involved ERK1/2 (Figure ) [52
], we reasoned that ERK1/2 could serve as an intermediary to connect IL-6 with ST3GAL5 expression. Accordingly, we tested the phosphorylation of ERK (Figure ) and found that pERK1/2 was inhibited by concentrations of IL-6 > 4.0 ng/ml (Figure ); the reduced ratio of pERK1/2 to ERK was consistent with dampened mRNA levels for the biosynthetic enzyme ST3GAL5 and the tandem up-regulation of the recycling enzyme NEU3 (Figure ). A noteworthy aspect of this study was that, although the effects of NEU3 and ST3GAL5 on "lubricating signaling pathways" [49
] have been previously evaluated separately, to our knowledge this is the first report where both enzymes were monitored simultaneously and found to respond to an external stimulus in a concerted manner that required transcriptional regulation of the biosynthetic and recycling enzymes in opposite directions.
Figure 7 Ganglioside metabolism and connections with ERK1/2. (A) This diagram shows the conversion of LacCer to the ganglioside GM3 by ST3GAL5 (and then to GD3 upon α-2,8-sialylation and to 9-O-AcGD3 upon O-acetylation); both GM3 and GD3 are converted (more ...)
The prolonged down-regulation of ganglioside GM3 upon IL-6 supplementation (Figure ) provides a two-pronged mechanistic explanation for long term attenuation of IL-6 and related responses in SMF-treated cells (for example, SMF-enhanced IL-6 levels returned to normal by day 6 (Figure ) followed by loss of growth inhibition by day 9 (Figure )). First, the loss of sialic acid – an important contributor to the carbohydrate-carbohydrate binding interactions that stabilize lipid assemblies [48
] – from GM3 can destabilize CD82-enriched microdomains [53
]. Assuming that the TLR4 receptor complex, which is also sensitive to the stability of its local microdomain environment [19
], responds to a reduction in GM3 levels in a similar manner, the signaling pathways activated by SMF over the first day or so of exposure could be 'turned off' by the loss of GM3 over longer time periods. A second mechanism to explain ganglioside-mediated attenuation of IL-6 can be postulated based on the findings by Müthing and colleagues that GSL such as GM3 increase Ca2+
flux through voltage gated channels [54
]. In an independent set of experiments, Yang and coworkers reported a strongly stimulatory effect for GM3 on the SR Ca2+-
]. Together, these findings indicate that the conversion of GM3 to LacCer in SMF-treated cells inhibits Ca2+
-dependent signaling pathways in a manner that attenuates the initial multi-pronged up-regulation of IL-6.
SMF regulates ganglioside production via NEU3 and ST3GAL5
The crosstalk between gangliosides and IL-6 (as summarized in Figures & and ), combined with the ability of SMF to modulate this cytokine (as shown by the data in Figure ), led us to consider whether SMF altered IL-6 via a ganglioside-mediated route (or vice versa). To investigate this possibility, NEU3 and ST3GAL5 – the enzymes that control the equilibrium between GM3 and LacCer (Figure ) and thus have the potential to indirectly modulate IL-6 (Figure &) – were monitored by qRT-PCR during the early stages of SMF exposure. In these experiments, up-regulation of NEU3 and inhibition of ST3GAL5 after one day of SMF exposure (Figure ) reminiscent of the effects of IL-6 supplementation (Figure ) were observed. Analysis of ganglioside levels in these cells showed that these transcriptional changes again worked in concert to decrease GM3 levels on the cell surface (Figure , top). A similar reduction in GM3 occurred in fixed and permeabilized cells where gangliosides situated in the secretory pathway are also measured (Figure , bottom). By testing both conditions, the possibility that surface changes merely reflected the redistribution of GM3 between the cell surface and intracellular compartments was discounted (this concern was raised by the hypothesis that SMF changes the biophysical properties of lipid bilayers thereby potentially affecting trafficking between surface and intracellular membranes). Interestingly, GD3 – which can modulate the biophysical properties of membrane raft assemblies similar to GM3 (and in essence serves as a reservoir for this monosialylated ganglioside, Figure ) – was also reduced by SMF (Figure ); this result can be explained by the ability of NEU3 to remove both sialic acid residues of GD3.
Figure 8 Effects of SMF on gangliosides GM3 and GD3 and IL-6. (A) mRNA levels increased for NEU3 and decreased for ST3GAL3 during the first 24 hours of SMF exposure consistent with a flow cytometry-measured reduction in GM3 (Panel B) and GD3/9-O-AcGD3 levels (the (more ...)
SMF regulates NEU3 and ST3GAL5 independently of IL-6
In order to gain insight into the cause and effect relationships that connect SMF, gangliosides, and IL-6, IL-6 was added to cells in the presence or absence of SMF. In this experiment IL-6 had the same effect on GM3 levels with or without concomitant magnetic exposure (Figure ). This result contrasted with the clear reduction in GM3 when IL-6 had been added to cells in the absence of SMF (as shown in Figure ). One explanation for these disparate results was that SMF activated a sequence of events where IL-6 transcription was first up-regulated leading to increased protein secretion, which in turn reduced GM3. This scenario, however, was discounted by a time course of NEU3 and ST3GAL5 mRNA expression over the first day of SMF exposure (Figure ) that showed that the transcriptional changes to these enzymes occurred before measurable IL-6 secretion took place (e.g., before 7 h, see Figure ). Therefore, SMF independently regulates IL-6 and gangliosides in a way that ultimately impinges on the same molecular mechanism (i.e., through NEU3 and ST3GAL5 transcription and activity). GM3 and GD3 also provide a putative explanation for the biphasic increase in IL-6 mRNA; at early time points a ganglioside-independent sequence of events (presumably involving, but not necessarily limited to, TLR4 activation or Ca2+ flux) occurs. As the initial signal fades, reduction of GM3 and GD3 could contribute to a second 'burst' of IL-6 expression by alleviating the suppressive effects of these gangliosides on IL-6 itself (Figure ) or on TLR4 (Figure ).
SMF – in combination with IL-6 – alters cell morphology
As described earlier, hEBD LVEC cells exposed to SMF experienced reduced proliferation without toxicity (Figure ), a response consistent with differentiation. To test if this phenomenon was linked to SMF or IL-6 production, cells were first treated with ≤ 4.0 ng/ml of IL-6 in the absence of SMF. IL-6 supplementation typically resulted in relatively minor (if any) change to cell morphology (Figure &). Occasionally, however, dendrite-like outgrowths reminiscent of neuronal cells developed in sub-populations of IL-6 treated cells (Panel C). By contrast, close to 100% of the cells attained distinctive morphology when SMF was combined with 4.0 ng/ml of IL-6 (Panel E; SMF alone had a much less pronounced impact on morphology, Panel D).
Figure 9 Morphological effects of SMF and IL-6 on hEBD LVEC cells. (A) Phase contrast images showing the normal morphology of ~90% confluent hEBD LVEC cells. (B) Slightly altered morphology typically was seen after incubating the cells with 4.0 ng/ml of IL-6 for (more ...)
One explanation for why both SMF and exogenous IL-6 supplementation was needed to elicit noticeable changes to cell morphology was that, because of the relatively small volume of cells (≤ 0.01%) compared to culture medium, any IL-6 secreted in response to SMF would be diluted ~10,000-fold. As a consequence, additional IL-6 supplementation was required to mimic levels achieved by comparable rates of IL-6 production in cells situated within an in vivo niche where the relative cell to interstitial volume ratios are much lower. Another (non-exclusive) explanation, supported by experiments where even 20 ng/ml IL-6 could not reproduce the combined effects of SMF plus 4 ng/ml IL-6 (data not shown), was that SMF-activated networks beyond IL-6 – such as those listed in Table – contributed to the morphological changes.
SMF promotes oligodendrocyte progenitor markers
To gain greater insight into the morphological changes induced in hEBD LVED cells by a combination of SMF and IL-6, we noted that IL-6 has a role in the regeneration of nervous tissue, usually promoting astrocyte formation [35
] and, accordingly, monitored the transcription of bone morphogenic protein 2 (BMP-2) and myelin basic protein (MBP) (Figure ). Interestingly, a decrease in mRNA for BMP-2, a protein that stimulates astrocytogenesis [59
], was observed suggesting that the hEBD cells were not differentiating into astrocytes as expected. To confirm this observation using immunofluorescent microscopy, no increase in the GFAP marker associated with astrocyte formation was observed in SMF and IL-6 treated cells (Figure ). Similarly, no increase was seen for NEF (Figure ), a marker associated with neuron differentiation.
Figure 10 Differentiation markers in SMF- and IL-6-treated hEBD LVEC cells. Test cells were incubated with 4.0 ng/ml IL-6 with concurrent SMF exposure (controls were incubated with neither) and the monolayers were co-stained with Oregon Green 488 phalloidin to (more ...)
Based on the lack of astrocyte or neuron differentiation, a third possibility was that the decrease in BMP-2 expression in SMF-treated cells removed the obstacle presented by bone morphogenetic proteins towards differentiation to oligodendrocyte lineages [60
]. Indeed, consistent with the decrease in BMP-2, an increase in myelin basic protein (MBP) transcription was observed (Figure ) providing a biochemical marker consistent with differentiation to an oligodendrocytes [61
]. Additional supporting evidence that SMF, combined with IL-6, leads toward oligodendrocyte progenitor formation was provided by the increased expression of vimentim (Figure ) and Gal-C (Figure ).
A timeline of SMF responses: Towards unraveling cause and effect relationships
The relationships between SMF, calcium, TLR4, gangliosides (and regulatory enzymes), MAPK pathway elements (p38 and ERK1/2) and IL-6 are outlined in Figure ; this diagram, however, does not provide a dynamic view that would provide insight into cause and effect relationships. Therefore, to summarize the time dependence of various aspects of hEBD LVEC cell responses to SMF exposure, early, intermediate, and longer term responses are summarized in Figure . During the first four hours (Panel A), changes to calcium flux occur within minutes as do MAPK responses (e.g., p38 phosphorylation, Figure ); effects on the transcription of IL-6, TLR4, NEU3, and ST3GAL5 mRNA lag slightly but show a strong response beginning between 2 and 4 hours. By contrast, secreted IL-6 remains unchanged. During the remainder of the first day of exposure (Panel B), mRNA levels of SMF treated cells trend back to control levels (the one exception is the 24 hour point for IL-6, which rebounds after a decline between 4 and 7 hours, this biphasic response mimics the impact of other stimuli on IL-6 [36
]). Also during the first day, while the impact of SMF on transcription of IL-6, TLR4, NEU3, and ST3GAL5 abates, phenotypic effects such as the accumulation of measurable levels of secreted IL-6 began to be manifest. In general, initiating events – for example, the impact of SMF on mRNA levels – were attenuated after the second day (as shown in Panel C) whereas "behavioral" responses (such as the secretion of IL-6 or the effects of SMF on proliferation) followed the same trend but lagged in time. During this multiday time period – while "intermediate" responses were returning to normal – long-lived changes to cell fate arose that included the morphological changes shown in Figure and the accumulation of pre-oligodendrocyte markers shown in Figure .
Figure 11 Timeline of SMF-induced, IL-6 associated responses in hEBD LVEC cells. Data is compiled from experiments reported throughout this paper to show (A) early responses that occur within four hours of the start of continuous SMF exposure, (B) intermediate (more ...)
Another lesson learned from the data shown in Figure was that SMF treatment set in motion a complex sequence of events that rapidly changed over time; consequently, the original goal of analyzing cellular responses by microarray profiling could have led to erroneous conclusions. For example, the complex and rapidly changing nature of IL-6 mRNA transcription could have led to the conclusion that the microarray results were simply irreproducible, as has been reported for much lower-strength fields [62
]. Alternately, the four and five day time points – where IL-6 mRNA levels were actually lower than controls – were not consistent with the strong, multifaceted up-regulation that occurred upon the initial exposure to magnetic stimulus thereby also providing misleading information if regarded in isolation. Therefore, we close by noting the benefits of the robust ability of software tools to uncover signature biological activity – namely, signaling responses associated with IL-6 – even when the key molecular player (i.e., IL-6) is undergoing rapidly changing or oscillatory behavior that would be difficult to understand by itself.