Relative SOD1 activity. Relative SOD2 activity. Relative SOD activity (Infected:Mock) + CP + DDC
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1 Supplementary Figure 1. SOD1 activity is significantly increased relative to SOD1 levels. SOD1 and SOD2 activities in the infected mork13 cells are shown normalised to their corresponding levels and relative to mock results. The plot shows that the significant SOD1 activity increase (t = 3.172; p =.33, n = 3) is not a result of increased levels. This suggests an allosteric modulator may be influencing SOD1 activity within the infected cells. SOD2 activity is reduced overall but SOD2 levels are also reduced, thus SOD2 activity relative to expression is unchanged. Relative SOD activity (:) * + CP Relative SOD1 activity + DDC Relative SOD2 activity
2 Supplementary Figure 2. Cleaved caspases 3 and 9 are found in the SR-VAD-fmk reactive aggregates. and infected mork13 cells were plated into chambered coverslips and allowed to grow to approximately 7% confluency. Cells were incubated with SR-VAD-fmk for 3 minutes before fixing and immunofluorescent labelling with (A) caspase 3 at a 1 in 4 dilution, (B) cleaved caspase 3 at a 1 in 4 dilution, (C) caspase 6/cleaved caspase 6 each at 1 in 5 dilution and (D) cleaved caspase 9 at a 1 in 5 dilution. All antibodies were purchased from Cell Signalling Technology (Genesearch, QLD, AUS). In merged images DAPI staining (blue) shows cell nuclei, green indicates the caspase antibody and red represents SR-VAD-fmk. Pro-caspase 3 shows little localisation to the SR- VAD-fmk reactive aggregates whereas cleaved caspase 3 shows staining within this area. Similar localisation is observed for caspase 9 but the aggregates appear devoid of pro- /caspase 6. Scale bars = 25 µm.
3 A. Pro-caspase 3 Caspase antibody SR-VAD-fmk Merge B. Caspase 3 C. Pro-/Caspase 6 D. Caspase 9
4 Supplementary Figure 3. ROS trapping or inhibition of caspases does not alter bcl-2 levels in prion infected cells. and infected mork13 cells were treated for 24 hours with (A) 5 µm TEMPO-9-AC radical trap or (B).6 µm SR-VAD-fmk caspase inhibitor and cells harvested for bcl-2 western blotting. Shown are representative bcl-2 blots with coomassie brilliant blue stain for loading. Data derived from three independent repeats is presented graphically (below) as a percentage of the untreated mock control value. Hollow bars indicate mock cell responses, filled bars represent infected cells with -/+ designating without/with treatments respectively. (A) Despite the mock cells treated with TEMPO-9-AC demonstrating increased levels of bcl-2 (t = 6.446, p =.23, n = 3), the infected cells did not show a response or recovery of bcl-2 levels upon treatment. (B) Caspase inhibition did not significantly influence bcl-2 level in either the mock or infected cells. A. B. TEMPO-9-Ac Bcl SR-VAD-fmk Bcl-2 stain stain 18kDa 18kDa Percentage mock control Percentage mock control
5 Supplementary Figure 4. Effect of chronic and acute hydrogen peroxide induced oxidative stress on SOD2 levels. To examine the effects of generic chronic oxidative stress insults on levels of SOD2 in cultured mork13 cells, cells were exposed to five doses of 1 µm hydrogen peroxide over five days then harvested for western blotting. In this time period no overt increase in cell death could be observed but a small increase is measurable by trypan blue staining (5% +/- 2.5). A single dose of 1 µm hydrogen peroxide was also applied overnight to a separate sample of cells to mimic an acute insult, with the cells harvested before most had died. Example western blots are show in (A) and quantification of SOD2 signal as compared with loading and relative to the untreated cell sample is shown in (B). SOD2 detection is significantly increased with chronic low-level oxidative stress caused by hydrogen peroxide (t = 4.586, p =.44, n = 3). Acute stress produced a highly variable detection of SOD, which is not significantly different from untreated cells (t =.765, p =.524, n = 3). A. B. 14kDa SOD2 stain for SOD2 (% Change from Untreated) Untreated * Chronic stress ns Acute stress
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