This Study Focused on the Effect Chloroquine in Astrocytes to Produce Interleukin-8 (IL-8)

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, IB and phosphor-IB (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.