Supplementary Data
Figure 1. TRAIL ligation increases intracellular ROS levels in human astrocytoma cells in a dose-dependent manner. (A) Cells were treated with varying doses of hrTRAIL for 30 min, and intracellular ROS levels were determined. *P ≤ 0.01 compared with the unstimulated sample. (B) Cells were incubated in the absence or presence of various ROS scavengers for 1 h and treated with hrTRAIL for an additional 4 h. Cell death was determined by FACS analysis after staining with tetramethylrhodamine ethyl ester (TMRE).
Figure 2. Generation of Cys6A-Caspase-3 mutant and BIAM labeling assay. (A) Sequencing data of the Cys6A-caspase-3 mutant construct. Mutated cysteine residues along with remaining ones are indicated. (B) Cells were transfected with either wild caspase-3 or C6A-caspase-3 contructs. After 24 h, cells were incubated in the absence or presence of NAC and/or z-VAD- fmk for 1 h and treated with hrTRAIL for an additional 1 h. Soluble lysates were labeled with
BIAM and immunoprecipitated with an anti-caspase-3 antibody (upper panel).
Figure 3. Suppression of NOX and PKC augments TRAIL-induced cell death. Cells were transfected with scrambled or PKC- or NOX4-specific siRNA for 48 h and treated with hrTRAIL (100 g/L) for various time periods. Cell death was measured by the MTT assay.
Figure 4. Hydrogen peroxide inhibits in vitro activity of recombinant caspase-3.
Recombinant caspase-3 (Abcam, Cambridge, MA, USA: 10 units) was incubated with various
doses (up to 100 mol/L) of H2O2 for 5 min in reaction buffer. Fluorogenic caspsase-3-specific
1 substrates were added for an additional 30 min at 37°C. Each value indicates mean values of 4 samples.
Figure 5. Specificity of PKC siRNA. Cells were transfected with various siRNAs for different
PKC isoforms (PKC-, and ) for 24 or 48 h. Cell lysates were subjected to immunoblot analysis for expression of PKC isoforms. Double stranded siRNA oligonucleotides specific for
PKC and PKC and antibodies against human PKC, PKC and -actin were obtained from
Santa Cruz Biotechnology.
Figure 6. Effect of NAC on TRAIL-induced NF-B activation. (A) Cells were incubated in the absence or presence of NAC (20 mmol/L) for 1 h and treated with hrTRAIL for various time periods. Cell lysates were subjected to immunoblot analysis using antibodies against IKK, phosphor-IKK, IB and phosphor-IB (Cell Signaling). (B) Cells were incubated in the absence or presence of NAC (20 mmol/L) for 1 h and treated with hrTRAIL for 6 h. Cells were washed with ice-cold PBS, and then RNA was extracted using a method based on guanidinium isothiocyanate phenol extraction, followed by ethanol precipitation. A linearized constructs for human MCP-1 and IL-8 (PharMingen) were in vitro transcribed with T7 RNA polymerase, resulting in antisense RNA probes. Ten g of total RNA were hybridized with riboprobes.
Values for each chemokine mRNA were normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA levels for each experimental condition. (C) Cells were incubated in the absence or presence of NAC (20 mmol/L) for 1 h and treated with hrTRAIL for varying time periods. Cell lysates were subjected to immunoblot analysis using antibodies against XIAP, c-
FLIPL (obtained from Cell Signaling) and -actin.
2 Figure 7. Critical role of PKC in Fas-induced apoptosis. (A) CRT-MG cells were incubated in the absence or presence of z-VAD-fmk for 1 h and then treated with an anti-Fas agonistic antibody CH-11 (500 g/L, Upstate, Lake Placid, NY, USA) for various time periods (0–5 h).
Soluble lysates (50 µg total protein/aliquot) were subjected to immunoblot analysis with an anti-
PKC antibody. Arrowheads indicate full-length (78 kDa) and cleaved (40 kDa) PKC fragments. The data shown are representative of two independent experiments. (B) Cells were transiently transfected with either scrambled control or PKC-specific siRNA for 48 h and treated with hrTRAIL or CH-11 for 2 h.
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