Time-Resolved FT-IR Microspectroscopy of Protein Aggregation Induced by Heat-Shock in Live Cells
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1 Time-Resolved FT-IR Microspectroscopy of Protein Aggregation Induced by Heat-Shock in Live Cells Elisa Mitri a,b, Saša Kenig a, Giovanna Coceano b, Diana E. Bedolla a, Massimo Tormen b, Gianluca Grenci b,c and Lisa Vaccari a* a) Elettra Sincrotrone Trieste, S.S. 14 km 163.5, Basovizza, Trieste, Italy b) IOM-CNR, TASC Laboratory, S.S. 14 km 163.5, Basovizza, Trieste, Italy c) MBI, National University of Singapore T-Lab, 5A Engineering Drive 1, Singapore * lisa.vaccari@elettra.eu 1
2 SPECTRAL FEATURES OF UNPERTURBED MCF-7 AND MDA-MB 231 CELL LINES Figure S1 shows the average of the second derivatives of subtracted spectra for all the sampled MCF-7 (red line) and MDA-MB 231 (blue line) heat-stressed cells under unperturbed conditions (t=0) in the regions cm -1 (a), cm -1 (b) and cm -1 (c). As already discussed in the manuscript, minimal differences can be detected among the two cell lines. Fig. S1: Second derivatives of water subtracted spectra averaged on all the MCF-7 (blue line) and MDA-MB 231 (red line) stressed sampled groups at t=0 min (unperturbed conditions). Shades are proportional to the standard deviation cm -1 (a), cm -1 (b) and cm -1 (c). Cells maintained at 37 ±0.1 C inside the microfluidic chamber for 2 hours do not display significant variations neither in the spectral shape as reported in figure S2 nor in the average integral values as highlighted in Table 1 and Table 2 of the paper. In Fig. 2 the mean spectrum of a representative Control group for both cell lines is plotted. Mean spectrum is obtained averaging all the spectra acquired at each experimental point for the representative group of cells considered. The thickness of the line is proportional to the standard deviation. As can be seen from the picture, for both cell lines there are only minimal variations that could be ascribed to the normal metabolic activity of cells. More importantly, there are not evidences of protein unfolding and/or aggregation. Fig. S2: Mean spectrum of the second derivatives for a representative group of MDA-MB 231 (a) and MCF-7 (b) control cells over all the experimental time in the spectral region between 1750 and 1400 cm -1. Line thickness is proportional to standard deviation. CELLS VIABILITY TEST Cell viability of both cell lines was verified by MTT and Trypan blue tests as described in the Experimental section. MTT results are reported in Fig. S3a and S4a, and related tables, for MDA-MB 231 and MCF-7 cells respectively. They demonstrate that cells retain cell viability even upon HS, since the cell viability is comparable with the one obtained for cells cultured at constant temperature of 37 C. Figure S3b and S3c show the results of the trypan blue assay performed on control and stressed MDA-MB 231 cells before the measurements, after two hours outside the incubator in a thermalized bath at the desired temperature and at the end of the experiment directly into the experimental chamber. The same results obtained for MCF-7 are instead reported in Fig. S4b and 4c respectively. 2
3 Fig. S3: Results of a MTT test represented as Absorbance of formazan at 570 nm for MDA-MB231 cells cultured at 37 ± 0.1 C (control) and at 42 ± 0.1 C (Stressed) (a); Optical images collected for the Trypan Blue test performed on control cells (b) before the measurements (Before), after 2 hours at 37 ± 0.5 C in a thermalized bath outside the incubator (2h Bath) and after 2 hours at 37 ± 0.5 C inside the experimental chamber (2h Device); Optical images collected for the Trypan Blue assay performed on stressed cells (d) before the measurements (Before), after 2 hours at 42 ± 0.5 C in a thermalized bath outside the incubator (2h Bath) and after 2 hours at 42 ± 0.5 C inside the experimental chamber (2h Device). The table in the figure summarizes the obtained results. Fig. S4: Results of a MTT test represented as Absorbance of formazan at 570 nm for MCF-7 cells cultured at 37 ± 0.1 C (control) and at 42 ± 0.1 C (Stressed) (a); Optical images collected for the Trypan Blue test performed on control cells (b) before the measurements (Before), after 2 hours at 37 ± 0.5 C in a thermalized bath outside the incubator (2h Bath) and after 2 hours at 37 ± 0.5 C inside the experimental chamber (2h Device); Optical images collected for the Trypan Blue assay performed on stressed cells (d) before the measurements (Before), after 2 hours at 42 ± 0.5 C in a thermalized bath outside the incubator (2h Bath) and after 2 hours at 42 ± 0.5 C inside the experimental chamber (2h Device). The table in the figure summarizes the obtained results. HSR OF MDA-MB 231 CELLS Analysis of HSR in MDA-MB 231 cells shows the existence of common response among all the sampled cell groups as previously described in the manuscript and supported by the inspection of the second derivatives and of the 2D plots (See Fig. S6a and S7a). The second derivatives and the Syn 2D plots display similar features among all the sampled groups; the Asyn 2D plots (reported in Fig. S5, S6c and S7c) slightly differ from each other due to the individual cellular susceptibility toward HS and the delay of 1 minute between each cellular spectrum. In all the Asyn plots the 1655 cm -1 component shows negative crosspeak with component at 1620 cm -1, but peculiar spectral details can be highlighted for each sampled group. 3
4 Fig. S5: Asynchronous 2D plot of the representative group of MDA-MB 231 cells within the first 40 min of HS application in the spectral region between 1750 and 1480 cm -1 (Amide I and Amide II bands). Specifically, the Asyn 2D plot of the representative group of MDA-MB 231 cells considered in the manuscript is reported in Fig. S5. From the analysis of the plot, it is possible to notice that four components of the Amide I band undergo major intensity variations at different experimental time points. These are centered at ~1655 (α-helix structures), ~1645 (random coiled/unordered structures), ~1635 (intramolecular β-structures) and ~1620 cm -1 (intermolecular β-structures). The correlations involving the components of the Amide II cannot be easily deduced due to the low intensity of the band and the consequent high influence of the spectral noise, inducing 2D peaks broadening. Amide I component at ~1655 cm -1 displays negative Asyn crosspeaks with the components at ~1645 cm -1 and ~1620 cm -1. Therefore, the event associated to the peak at ~1655 cm -1 occurs later than the one related to the one at ~1645 cm -1 and before the one at ~1620 cm -1. Among the 1645 cm -1 and 1620 cm -1 components, a negative crosspeak is seen. A defined Syn crosspeak could not be detected for these two peaks, since it is partially masked by the strongest crosspeak between 1655 and 1620 cm -1 components. However, it is located in a negative region of the Syn plot and therefore, according to Noda, the event related to the 1645 cm -1 component should precede the one related to the 1620 cm -1 one. The observation of a negative Asyn crosspeak between the signals at ~1635 cm -1 and ~1620 cm -1, correlated by a negative Syn crosspeak, suggests that the events related to the lower wavenumber signal occurs after the ones associated to the ~1635 cm -1 component. Finally, the component at 1635 cm -1 has a negative crosspeak with the component at 1645 cm -1. A defined Syn crosspeak could not be seen for these two peaks that however stands in a positive region of the Syn plot. Therefore, following the same reasoning as before, the event related to the 1645 cm -1 component should precede the one related to the 1635 cm -1 component. 4
5 Fig. S6: Second derivatives of a group of MDA-MB 231 cells at different sampled times within 2 hours of HS application in the spectral region between 1750 and 1480 cm -1 (a), Synchronous (b) and Asynchronous (c) 2D correlation plots of absorbance spectra for the same group of MDA-MB 231 cells within the first 40 min of HS application in the spectral region between 1750 and 1480 cm-1 (Amide I and Amide II bands). 5
6 Fig. S7: Second derivatives of a group of MDA-MB 231 cells at different sampled times within 2 hours of HS application in the spectral region between 1750 and 1480 cm -1 (a), Synchronous (b) and Asynchronous (c) 2D correlation plots of absorbance spectra for the same group of MDA-MB 231 cells within the first 40 min of HS application in the spectral region between 1750 and 1480 cm-1 (Amide I and Amide II bands). Figure S8 plots the second derivatives of the spectra in the regions of stretching and bending modes of methyl and methylene moieties ( cm -1 and cm -1 ) for the three selected groups of MDA-MB 231 cells at different experimental times. Fig. S8: Second derivatives of the spectra of the representative groups of MDA-MB 231 cells considered in the manuscript (a), of the cell group in Fig. S6 (b) and of the one in Fig. S7 (c) in the in the spectral regions of stretching and bending modes of methyl and methylene moieties ( cm -1 and cm -1 ), at different sampled times within 2 hours of HS. 6
7 HSR OF MCF-7 CELLS The HSR of MCF-7 cells have been investigated following the same approach used for MDA-MB 231 cells. Figure 2 in the manuscript shows the second derivative spectra as well as Synchronous 2D correlation plot of the chosen representative group of MCF-7 cells. Fig. S10 and S11 report the results for other two MCF-7 cell groups. With respect to the evolution of the cellular protein structures in HSR, all the sampled MCF-7 cell groups elicit late HSRs comparable to MDA-MB 231 cells and also share common features of the final state, characterized by the ultimate accumulation of extended β-aggregates. In the following, we will highlight the similarities drawn out especially from derivatives and Syn 2D plots. In Fig. 2a of the manuscript, the 1D time-evolution of second derivatives of the representative cell group in the 1700 and 1480 cm -1 spectral region is plotted. The Amide I band, peaked at 1652 cm -1, is broadened toward lower wavenumbers at t=0 minutes (red curve in Fig. 2a), and one shoulder can be identified at 1681 cm -1. Once applied the HS (brown curve in Fig. 2a), the broadening of the Amide I band is even more pronounced and a second component centered at 1641 cm -1 becomes apparent. At t=10-15 minutes (orange and yellow curves in Fig. 2a), a partial reshaping of the Amide I band in a state closer to the one detected at t=0 can be seen, as well as the presence of a signal centered at 1627 cm -1. At t=20 min (light green curve in Fig 2a), a clear component centered at 1620 cm -1 is first detected. Between 25 and 40 minutes, the spectral profiles fluctuates through states characterized by a different degree of broadening of the 1652 cm -1 component, and the alternation of the 1630 or 1620 cm -1 shoulders, as well as the presence of both of them. The steady presence of β- aggregates characterizes the long-term HSR of MCF-7 cells of the representative cell group (blue curve in Fig. 2a), as well as of all the other sampled groups, as already highlighted. At the end of the experiment (black curve in Fig. 2a), the presence of β- aggregates is even more pronounced, and, for the specific case, an outstanding broadening of the Amide I band can be appreciated. This feature is common to all other sampled groups (see as an example Fig. S10a and S11a). Concerning the 2D plots of absorbance spectra in the Amides region, both Syn and Asyn plots of MCF-7 cells present autopeaks and crosspeaks generally more broaden than MDA-MB 231 cells. The Amide I band component centered at 1620 cm -1 dominates the Syn plots of all MCF-7 sampled cells (see Fig. 2b and Fig.S10b). In Fig. S11b is reported the Syn plot of the unique cell group for which the most intense autopeak is centered at ~1645 cm -1. Autopeaks with lower intensity were occasionally detected for the spectral component centered at ~1655 cm -1, the one most pronounced in the second derivative average spectrum. Other autopeaks for the Amide II components centered at ~1555 and/or at ~1525 cm -1 could be detected for all cell groups. The spectral component at 1620 cm -1 is always negatively correlated with the ones in the cm -1 spectral region and with the one centered at 1555 cm -1, revealing an opposite variation, while a positive crosspeak relates the 1620 cm -1 component with the one at 1525 cm -1, that therefore follows the same trend. Finally, the crosspeak between the spectral components in the cm -1 is positive with the 1555 cm -1, while negative with the 1525 cm -1 contribution. Summarizing, the two spectral components at 1620 and 1525 cm -1 follow a similar trend, opposite to the one taken by all the other spectral components. For what concerns the Asyn plot of the representative group of MCF-7 cells (Fig. S9), a well-defined negative crosspeak between the spectral component centered at 1635 cm -1 and 1620 cm -1 can be seen. However, since in the corresponding Syn plot the correlation intensity vanishes, nothing can be told about the time correlation of the events related to those bands. A weaker positive crosspeak is detected in the region between 1655 and 1635 cm -1, but an unambiguous interpretation is not possible due to the peak broad nature. Asyn plots of MCF-7 cells in Fig. S10c and S11c are also difficult to interpret, therefore the sequence of the events characterizing the immediate and early HSR in MCF-7 cells can be better followed by focusing on the time evolution of the second derivatives of the spectra. Fig. S9: Asynchronous 2D plot of the representative group of MDA-MB 231 cells within the first 40 min of HS application in the spectral region between 1750 and 1480 cm -1 (Amide I and Amide II bands). 7
8 Fig. S10: Second derivatives of a group of MCF-7 cells at different sampled times within 2 hours of HS application in the spectral region between 1750 and 1480 cm -1 (a), Synchronous (b) and Asynchronous (c) 2D correlation plots of absorbance spectra for the same group of MCF-7 cells within the first 40 min of HS application in the spectral region between 1750 and 1480 cm -1 (Amide I and Amide II bands) 8
9 Fig. S11: Second derivatives of a group of MCF-7 cells at different sampled times within 2 hours of HS application in the spectral region between 1750 and 1480 cm -1 (a), Synchronous (b) and Asynchronous (c) 2D correlation plots of absorbance spectra for the same group of MCF-7 cells within the first 40 min of HS application in the spectral region between 1750 and 1480 cm -1 (Amide I and Amide II bands). Figure S12 plots the second derivatives of the spectra in the regions of stretching and bending modes of methyl and methylene moieties ( cm -1 and cm -1 ) for the three selected groups of MCf-7 cells at different experimental times. Fig. S12: Second derivatives of the spectra of the representative groups of MCF-7 cells considered in the manuscript (a), of the cell group in Fig. S10 (b) and of the one in Fig. S11 (c) in the in the spectral regions of stretching and bending modes of methyl and methylene moieties ( cm -1 and cm -1 ), at different sampled times within 2 hours of HS. 9
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