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Blood, Vol. 112, Issue 13, 5219-5227, December 15, 2008
Cell functions impaired by frataxin deficiency are restored by drug-mediated iron relocation
Blood Kakhlon et al.
112: 5219
Supplemental materials for: Kakhlon et al
Files in this Data Supplement:
- Figure S1. DHR-based measurement of the mitochondrial labile iron pool (JPG, 67.2 KB)
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Trex cells induced for 6 days with tetracycline were labeled with dihydrorhodamine123 and fluorescence was monitored microscopically before (0′) and 20′ and 30′ after addition of 50 µM H2O2. DFR was added at 20′ for the last 10′ as indicated (lower right image). Representative fluorescence images are shown in the left panel and estimated fluorescence intensities (relative to the basal fluorescence at 0′) are shown in the right panel. Fluorescence intensity values were calculated as means of 5 ROI (n=5 independent experiments). *, significant difference (p < 0.05) in one-tailed, paired t-tests. Scale bar= 10 µm.
- Figure S2. The effects of DFR and SIH on the mitochondrial labile iron pool (JPG, 52.8 KB)
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Mitochondrial LIP was measured in DHR-loaded Trex cells induced with tetracycline (1 µg/ml) for 6d (Tet+), exposed overnight to respective chelator DFR or SIH (each at 50 µM) and subsequently to 50 µM H2O2 for 20′. H2O2 induced a significant raise in fluorescence only in cells (labeled Tet+) not treated with either chelator (n=3, p < 0.05, one-tailed paired t-test).
- Figure S3. DFP affects the recovery of aconitase activity from iron depletion (JPG, 41.2 KB)
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Trex cells induced (Tet+) or not (−) with tetracycline for 6d were treated for 30h with 150 µM DFO, washed, and re-cultured for 3h in full medium with or without DFP. Cells were subsequently lysed and lysates were subjected to in-gel aconitase activity assays. The Tet+ system with no DFP supplementation was the only one that showed no significant increase following recovery (n=3, p<0.05, one-tailed paired t-tests).
- Figure S4. The effect of DFP on aconitase activity (JGP, 12 KB)
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Trex cells (uninduced) were either lysed before treatment (C), treated for 30 h with 150 µM DFP and then lysed (DFP), or treated with 150 µM DFP for 30 h washed, and re-cultured for 3h in full medium (3h rec.). Subsequent to lysis, lysates (50 µg protein) were subjected to both in-gel aconitase activity assay (upper panel) and immunobloting (lower panel) with Ab against mitochondrial aconitase.
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