To supplementary Supplementary data Figure 1. Reduced expression of rpS6 shows resistance to TRAIL in SKHep-1 cells. Wild type and SKHep-1 rpS6 knock-down cells (SKHep-1 sh-S6 R7 #1D) were incubated with 75 ng/ml TRAIL for the indicated times. After stained with Annexin V-PE and 7-AAD (Pharmigen #559763), the cells were analyzed with FACScaliburTM. Cells stained with Annexin V-PE and 7-AAD are indicated (%) and apoptotic. Supplementary data Figure 2. Western blotting showing the expression level of GFP-fused rpS6 and endogenous rpS6. HeLa cells were transfected with control (GFP) and GFP-fused S6 (GFP-S6). After 24 h, cell extracts were prepared and analyzed with Western blotting using anti-rpS6 antibody. Endogenous rpS6 (Endo. rpS6) is shown. Supplementary data Figure 3. S6K1 induces rpS6 phosphorylation and suppresses TRAIL-induced apoptosis. (a) HeLa cells were transfected with GFP and either pcDNA, S6K1 (WT), or S6K1 T389E constitutive active mutant for 24 h. Cells were then exposed to TRAIL (35 ng/ml) in the presence or absence of serum for the indicated times. Cell death was determined based on the morphology of GFP-positive cells (lower panel). Cell extracts were examined with Western blotting using anti-rpS6 and antiphospho-rpS6 antibodies (upper panel). (b) HeLa cells were pre-treated with rapamycin (200 ng/ml) for 24 h and then left untreated or exposed to TRAIL (50 ng/ml) for 3 h or TNF- (30 ng/ml) with cycloheximide (CHX, 5 g/ml) for 3 h. Cell death was examined under a fluorescence microscope after staining with ethidium homodimer-1. Bars represent mean SD (n = 4). Supplementary data Figure 4. RT-PCR analysis showing the reduced level of DR4 mRNA in SKHep-1 rpS6 knock-down cells. Total RNA was extracted from wild type (Control) and rpS6 knock-down cells (GFP-AS-S6 #3 and sh-S6 R7 #1D) and then analyzed by RT-PCR using gene-specific synthetic oligonucleotides. Supplementary data Figure 5. Deletion mapping for pro-apoptotic activity of rpS6. (a) Schematic diagram of rpS6 deletion mutants (left panel). HeLa cells were transfected with EGFP, EGFP-rpS6, EGFP-rpS6 ΔA, or EGFP-rpS6 ΔB (GFP, GFP-S6, GPF-S6 ΔA, and GFP-S6 ΔB) for 24 h and analyzed for the expression levels with Western blotting using anti-GFP antibody (right panel). NS indicates non-specific bands. (b) After pre-treatment with 200 ng/ml rapamycin (Rapa.) or DMSO for 12 h, HeLa cells were transfected with EGFP, EGFP-S6, EGFP-S6 ΔA, or EGFP-S6 ΔB for 24 h and then incubated with TRAIL (100 ng/ml) in the presence or absence of rapamycin (200 ng/ml). Cell viability was determined by counting GFP-positive cells showing condensed and fragmented nuclei after staining with Hoechst 33258. Bars represent mean SD (n = 3). Methods Construction of rpS6 deletion The cDNAs containing nucleotide sequence 1-210 and 1-450 of human rpS6 were amplified by PCR using synthetic oligonucleotides - forward primer 5' - CGG AAT TCC GAT GAA GCT GAA CAT CTC CTT CCC A - 3′ and reverse primer 5′- CGG GAT CCC GAT GGG TCA AGA CAC CCT GCT TCA T - 3′ for GFP-S6 ΔA (1-210), or reverse primer 5′- CGG GAT CCC GTT CTT TAG AGA GAT TGA AAA GTT T - 3′ for GFP-S6 ΔB (1-450). The PCR products were subcloned into pEGFP (Clontech). RT-PCR analysis The experimental procedures were described in Material and methods. Synthetic oligonucleotides were used as primer for PCR: DR4 forward primer 5'- CTC GGC TCC GGG TCC ACA AG- 3′ and reverse primer 5'- CAC CCT CTG CTG CAC TTC C- 3′; DR5 forward primer 5'-ATG GAA CAA CGG GGA CAG- 3′ and reverse primer 5'CTT GGA CAT GGC AGA GTC- 3′; caspase-8 forward primer 5'-ATG GAC TTC AGC AGA AAT CTT TAT GAT A- 3′ and reverse primer 5'-GGC AGA AAT TTG AGC CCT GCC TGG TGT C- 3′; caspase-9 forward primer 5'-ATG GAC GAA GCG GAT CGG CGG CTC CTG C- 3′ and reverse primer 5'-GTC CAC TGG TCT GGG TGT TTC CGG TCT G-3′. Apoptosis assay For annexin V-PE and 7-AAD staining, cells were stained by annexin V-PE and 7-AAD (Pharmingen # 559763, San Diego, CA) and cell death rates were examined by annexin V-PE and 7-AAD positive cells with FACScaliburTM.