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.