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Blood, Vol. 109, Issue 7, 2903-2911, April 1, 2007
Disease-associated mutations in CIAS1 induce cathepsin B–dependent rapid cell death of human THP-1 monocytic cells
Blood Fujisawa et al.
109: 2903
Supplemental materials for: Fujisawa et al, Vol 109, Issue 7, 2903-2911
Files in this Data Supplement:
- Figure S1. The rapid cell death induced by the disease-associated mutant of CIAS1 is not due to the epitope tag (PDF, 111 KB) -
WT-CIAS1 and Y570C-mutants of CIAS1 without an epitope tag were cloned into the pIRES2-EGFP vector. Empty vector (0.5 µg) and the plasmid encoding the WT-CIAS1 or Y570C mutant were introduced into 1 × 106 THP-1 cells. Three hours after transfection, cells were analyzed by flow cytometry. Cell death was assessed by examining for annexin V– and 7-AAD–positive staining. Representative data from 3 independent analyses that gave similar results are shown.
- Figure S2. Disease-associated mutants of CIAS1 also induce lysosomal leakage and mitochondrial damage (PDF, 173 KB) -
The plasmid encoding WT-CIAS1 (0.5 µg) or 1 of the disease-associated mutants (R260W, D303N, or Y570C) was introduced into 1 × 106 THP-1 cells. Three hours after transfection, cells were analyzed by flow cytometry. Cell death was assessed by examining for annexin V– and 7-AAD–positive staining (top panels). Ss LL was assessed by incubating cells with 1 µg/mL acridine orange. SsLL represents the loss of red fluorescence, and the percentage loss of red fluorescence was calculated in the region under the bar (middle panels).  m was assessed by incubating cells with 1 µM MitoTracker DeepRed 633. m is represented as the loss of deep red fluorescence (bottom panels). The levels of both ssLL and m induced by each mutation were compatible with the degree of cell death as well as ASC-dependent NF- B activation (Figure 1B). Representative data from 3 independent analyses that give similar results are shown.
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