Supporting Information An Electrochemical System for the Study of Trans-Plasma Membrane Electron Transport in Whole Eukaryotic Cells

The study of trans-plasma membrane electron transport (tPMET) in oncogenic systems is paramount to the further understanding of cancer biology. The current literature provides methodology to study these systems that hinges upon mitochondrial knockout genotypes in conjunction with cell surface oxygen consumption, or the detection of an electron acceptor using colorimetric methods. However, when using an iron redox based system to probe tPMET, there is yet to be a method that allows for the simultaneous quantification of iron redox states while providing an exceptional level of sensitivity. Developing a method to simultaneously analyze the redox state of a reporter molecule would give advantages in probing the underlying biology. Herein, we present an electrochemical based method that allows for the quantification of both ferricyanide and ferrocyanide redox states to a highly sensitive degree. We have applied this system to a novel application of assessing oncogenic cell-driven iron reduction and have shown that it can effectively quantitate and identify differences in iron reduction capability of three lung epithelial cell lines.


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Method S-1. Stability of FIC in cell culture conditions. 0.01 mM FIC and FOC (Acros Organics) solutions were both made in Hanks' Balanced Salt Solution (HBSS). The two solutions were mixed in a 1:1 ratio to give a 0.01 mM solution containing 0.005 mM FIC and 0.005 mM FOC. The resulting solution was subject to linear sweep voltammetry, as de-scribed above, with and without incubation for 2 hours at 37°C, 5% CO2. All parameters and procedures for electro-chemical analysis were as outlined for the section on calibration procedure. HBSS was processed in the same way as tested samples and values obtained subtracted for normalisation purpose.  Solutions were incubated at 37˚C in 5% CO 2 atmosphere for 2 hours (red) and no incubation at room temperature and atmospheric conditions for 2 hours (black). Linear sweep voltammetric analysis was performed at a starting potential of 500 mV and an end potential of -150 mV, at a scan rate of 10 mV s -1 . Pseudo-steady state anodic current values were used to calculate FOC concentration (Right panel, crossed bar), and pseudo-steady state cathodic values used to calculate FIC concentration (Right panel, white bars). Total iron concentration (Right panel, whole bar) was calculated by adding FOC and FIC together. The right panel depicts total iron concentration for before and after incubation at 37˚C in 5% CO 2 , each total concentration bar also demonstrates the amount of FIC and FOC for before and after incubation, represented as parts of the total iron concentration. Paired t-test indicated no significant difference between scans before and after incubation, p= 0.363. Error bars show ± 1SD from the mean. N=3, n=3.
The stability of FIC and FOC at our defined cell culture conditions, over two hours, needed to be determined. This was done to ensure that both redox states of iron did not precipitate out of solution, or degrade, in the conditions tested, which would alter our total iron concentration. This involved testing a non-incubated and incubated sample of a 1:1 mixture of FIC (0.005 mM) and FOC (0.005 mM). This experiment also demonstrates our ability to simultaneously detect two iron redox states, a major advantage of our application. Figure 1 demonstrates that there is no change in the current values between curves in the voltammogram when our iron mixture is subjected to heat and increased CO 2, in line with the conditions during cell experiments. The equations from Figure 2 were used to calculate concentrations of FIC (bottom equation) and FOC (top equation), and these concentrations added to determine our total iron concentration. Paired two-tailed t-test showed no significance between the total iron concentrations calculated at p = 0.289, meaning that our data indicates that there is no iron degradation or precipitation when subjected to the conditions tested. We can see our total iron concentration is also near 10 µM, which indicates our system is valid.
Method S-2. Investigation of electrode fouling. 0.01 mM FIC (Acros Organics) solution was made in Phosphate Buffer Saline (PBS). The resulting solution was subject to linear sweep voltammetry, at 37˚C with a starting potential of 600 mV and an end potential of -250 mV, at a scan rate of 10 mV s -1 . A cell-incubated FIC solution was then tested and immediately after the same initial FIC solution (tested before cell-incubated sample) was re-tested. Total iron concentration as calculated by adding FOC and FIC concentrations from anodic and cathodic current values. PBS was processed in the same way as tested samples and values obtained subtracted for normalization purpose.
Method S-3. Bicinchoninic acid assay. Following removal of the supernatant for electrochemical analysis of cellconditioned samples, each well was washed three times for 5 minutes each with PBS. 1 ml of 2% triton X-100 solution was then added to each well and the plates incubated at 37°C for 20 minutes. Following this the lysed celltriton samples were transferred to a microcentrifuge tube and centrifuged at 16000g for 20 minutes. The supernatant was removed and transferred to a fresh microcentrifuge tube before analysis using a Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific Ltd) and the manufactures recommended protocol.    Calibration curve for potassium ferrocyanide (FOC) and potassium ferricyanide (FIC) for concentrations between 0 and 10 µM, with 95% prediction bands shown (dotted lines). FOC and FIC were made in Hank's Balanced Salt Solution (HBSS). Linear sweep voltammetric analysis was performed at a starting potential of 500 mV and an end potential of -150 mV, at a scan rate of 10 mV s -1 , and at 37˚C. GraphPad Prism 7.01 software was used to calculate linear regression lines in addition to 95% prediction bands, which are expected to encompass 95% of future data points. SD error bars are also shown for all points. N=3, n=3.  (PBS), tested before and after the microelectrode had been used with cell-incubated material. Linear sweep voltammetric analysis was performed at a starting potential of 600 mV and an end potential of -250 mV, at a scan rate of 10 mV s -1 , 37 ˚C. Pseudo-steady state anodic current values were used to calculate FOC concentration and pseudo-steady state cathodic values used to calculate FIC concentration. Total iron concentration was calculated by adding FOC and FIC together. The FIC solution was tested before (Right panel, dotted bar) and after (right panel, crossed bar) the electrode has been used with cell-incubated material. Paired t-test indicated no significant difference between scans before and after cell-related electrode use. (p=0.1406). Error bars show ± 1SD from the mean. N=9, n=3.
It was important to determine whether the electrode was likely to be fouled in salt based buffer when working with cell solutions. This involved testing a 0.01 mM FIC solution before and after the electrode was used with cellincubated buffer. We found there to be no change in the concentration of iron before and after testing, and therefore conclude that there was no element of electrode fouling present. This is interesting as it addresses one of the potential reasons for the reduction in total iron concentration that we see in our cell-induced iron reduction experiments. Figure 3A. Assessment of growth rate using a Tecan plate reader with cell counting and viability functions.

Method S-4 Relating to
Assessment of growth rate was done using a Tecan plate reader with cell counting and viability functions. To optimise this technique the range of cell sizes acknowledged by the machine had to be altered for each cell type. Due to Calu-3 and H1299 cells being larger than their A549 counterparts the range selected was from 14-28 µm, whereas for A549 cells this range was set at 10-24 µm. Seeding density was also subject to variation between cell lines, to ensure a growth curve with three distinct areas which include the lag, exponential and a final plateau phase that mimics the initial lag phase. Harvesting was within the exponential phase as the cells would have the highest metabolic rate in this region, and we hypothesised they would therefore reduce more iron. Experiments indicated that Calu-3 cells would not grow correctly when seeded at 150,000 cells/well in a 12 well plate, producing multiple plateau phases, this is illustrated in Figure S-2. This was increased to 250,000 cells/well in a 12 well plate, which produced three distinct regions as mentioned. In contrast, H1299 and A549 cells achieved the same growth curve structure when seeded at 100,000 cells/well in a 12 well plate. The importance of this is as an indication of the cells inherent proliferative ability.  Growth rate study for Calu-3 cells. Calu-3 cells were seeded at 150'000 cells/well in a 12 well plate and grown for 8 days. Viability was tested by tryphan blue exclusion and viable cell number counted using a Tecan microplate reader (Tecan Ltd, Weymouth, UK). 150'000 cells/well seeding density yielded multiple growth sections, with a central plateau phase. Seeding density was increased to yield three distinct growth phases ( Figure  3). N=1, n=3.  LDH assay data is shown in Figure S-6A. Dulbecco's Modified Eagle Medium (DMEM) was selected as the negative control to mimic ideal conditions, and assigned 0% LDH release. Triton X-100, a well-used detergent, was used as positive control to represent 100% cell death and therefore maximum signal, it was assigned 100% LDH release. For both A549 and H1299 cells HBSS caused a lower rate of membrane perturbation at significance of p = 0.011 and p = 0.003, respectively. Calu-3 cells showed no preference for PBS or HBSS at p = 0.230. As a result of these experiments HBSS was chosen as the supporting electrolyte. The rationale behind this decision was that if PBS was causing a higher incidence of membrane perturbation there would likely be a higher incidence of intracellular species that were electrochemically active released into the supporting electrolyte. In addition, this would be more likely to cause electrode fouling. Thus, choosing HBSS would ensure there was less electrochemical interference with the electrochemical system, and there would also be a lower incidence of cell death. It was important to establish iron was having no effect on cell viability.

S-5
Having selected our supporting electrolyte, FIC ability to induce cytotoxicity on each cell line was investigated using the MTS assay. The other reason for choosing this assay was to provide values for the mitochondrial metabolic rate of our cell lines. This would allow us to test the hypothesis that tPMET systems are used in cells with high metabolic rates as a means to aid in the regeneration of NAD + and facilitate increased rates of glycolysis 3 . We wanted to investigate this as the electron transport chain (ETC) within the mitochondria is involved with NAD + regeneration by feeding electrons from NADH into the ETC. DMEM was selected as negative control to mimic ideal conditions, and assigned 100% cell viability. Triton X-100 was used as positive control to represent 100% cell death, and assigned 0% viability. A HBSS control was also set up to ensure we could test the metabolic rate effects of HBSS against ideal conditions using DMEM. As can be seen in Figure S-6B there is no effect upon relative metabolic rate until a concentration of 10 mM FIC is reached. As a result of this investigation a concentration of 0.01 Mm FIC was selected for subsequent experiments. This ensured the chosen concentration was well within the non-cytotoxic range, but also meant a higher signal-to-noise ratio was more likely, as any reduced iron would constitute a larger portion of the overall iron concentration if the total concentration was smaller. Interesting results were achieved when comparing HBSS relative metabolic activities to DMEM controls (100%). For Calu-3 cells there was no cytotoxic effect at p = 0.696, whereas H1299 and A549 cells were affected at p = 0.0002 and p = 0.002, respectively. This provides an insight into what is causing cytotoxic effects in our system. For Calu-3 cells it appears to be inherent viability deficit even in ideal conditions, whereas H1299 and A549 appear to be viable in ideal conditions and undergo stress when transferred into an alternate medium.
By taking the absorbance values in DMEM conditions for all cell types, and normalising using the number of cells from our growth study we could gain some information about the cells mitochondrial metabolic rate. Interestingly, Calu-3 cells had the highest normalised absorbance (and hence mitochondrial metabolic rate) of 9.56 x 10 -5 A.U. per cell, whilst A549 cells were similar at 9.23 x 10 -5 A.U. per cell, and H1229 significantly different to both at 7.16 x 10 -5 A.U. per cell (p = 0.0001 for A549/Calu-3 cells vs H1299 cells).
It was important to look at the pH changes when incubating with cells as there were noticeable changes in the voltammograms when using cells to reduce iron. There is a pH change for HBSS buffer (with or without FIC) when incubated with all cell lines at p < 0.0001. For HBSS the pH when non-incubated is 7.62, which drops to 7.17, 6.95 and 6.95 for Calu-3, H1299 and A549 respectively. This is a small change for both HBSS of 0.45 pH for Calu-3 cells and 0.67 pH for both H1299 and A549. For FIC the pH when non-incubated is 7.62, which drops to 7.13, 7.11 and 7.05 for Calu-3, H1299 and A549 cells respectively. This is again a small change of 0.49, 0.51 and 0.57 respectively. These small changes that we see may be enough to slightly alter the conditions at the electrode surface and thus cause the 'flattening' of the voltammogram from the altered half wave potential. There is a difference in pH for some cell lines when comparing between HBSS and FIC incubated with the same cells for two hours. For HBSS and FIC only (no incubation) there was no change at p >0.9999. For Calu-3 cells there was no significant difference at 0.7806, for H1299 there was a difference at p = 0.0003, and also for A549 at p = 0.0310. This also could contribute to changes in our voltammograms as the baseline subtraction may not be completely aligned to the sample. However, this does not affect our results as by using the first derivative method of assessing the pseudo-steady state we are able to accurately pinpoint the relevant voltages for steady states despite changes to the half-wave potential or voltammogram shape.

Method S-6. Inductively coupled plasma mass spectrometry (ICP-MS).
Cells were plated and grown as described for cellular electrochemistry experiments, the incubation with HBSS and FIC for two hours (as described in the cellular electrochemistry section). A549 cells were passage 13-25, H1299 passage 14-29 and Calu-3 passage 32-40. Once supernatant had been removed cells were washed four times for 5 minutes each time with warm PBS, to ensure complete removal of any FIC solution. Cells were then desorbed using trypsin, and counted as outlined previously for the growth study. Centrifugation at 200g for 5 minutes resulted in cell pellets to which 300µl of RIPA lysis buffer was then added, and thoroughly mixed with cells for 45 mins. The resulting suspension was then frozen at -20°C for 2 hours, and then thawed to ensure complete lysis. This cycle was repeated three times. Thawed centrifuge tubes then had 1ml of concentrated (12M) HCl acid added to the cell lysate, and were left for six hours to dissolve the iron content of the cell/debris suspension. 5ml of deionized water was then added and the suspension was centrifuged at 2500g for 15 minutes. The resulting supernatant was transferred to a new centrifuge tube and analyzed using ICP-MS.
S-8  Two-way ANOVA analysis with Sidak's multiple comparison test shows no significance difference in iron content between treatments with HBSS and FIC. Additionally no significance difference is observed in iron content between cell types. SEM error bars shown. N=3, n=3