Supplementary files
Methods and legends
Reagents and Antibodies:
Human recombinant IL-2 (Proleukin®) was obtained from CHIRON (Emeryville, CA), human recombinant insulin and LY294002 were obtained from Sigma Aldrich ( Milan, Italy). Sgk-1 specific and scrambled siRNAs were supperplied from Upstate (Segrate,MilanItaly), and used according to the manufacturer’s instructions. Antibodies to phospho-Ser422 Sgk1 (rabbit), phospho-Ser422 Sgk1 (goat), Sgk1 (goat), phospho-ERK1/2 (mouse), ERK1/2 (rabbit), Caspase 8 (rabbit), Shc-1 C-20 (rabbit), IL-2R -chain( rabbit), C-myc (rabbit and mouse) and anti -tubulin (rabbit) were purchased from Santa Cruz (Santa Cruz Biotechnology, Santa Cruz, CA). The antibody specific for Sgk1 (rabbit) and the specific Sgk1 peptide (399-412, CGKSPDSVLVTASVK) used to raise the antibody in rabbit, and phospho-tyrosine (mouse) were purchased from Upstate, and the antibody specific to CD25-FITC conjugate was obtained from Serotec UK.
Recombinant DNAs:
The coding sequences of myc-tagged wild-type Sgk1 and dominant negative D222ASgk1 within pCINeo, were separately subcloned into the EcoRI, NotI and EcoRI, XhoI sites of the PcDNA4TO expression vector (Invitrogen, Milan,Italy S.r.l.), respectively. The dominant negative Sgk1 was obtained through site directed mutagenesis as previously described 12. Both constructs gave higher expression in COS-7 cells relative to the original pCINeo constructs (data not shown).
Cell culture:
A-498 ( epithelial kidney cancer cells) and HEK293T (human embryonic kidney) cells were grown in DMEM (Invitrogen), supperplemented with 10% FBS (Invitrogen s.r.l.), and antibiotics (penicillin, 100 units/ml, and streptomycin, 100g/ml) (Gibco-Invitrogen), in 5% CO2 in a humidified incubator. Jurkat (T-cell leukemia/lymphoma) and IM-9 (B-cell lymphoma) were grown in RPMI (Invitrogen), supperplemented with 10% FBS(Gibco) and antibiotics (penicillin, 100 units/ml, and streptomycin, 100g/ml) (Gibco) in 5% CO2 in a humidified incubator.
Solubilization of cells
Cells were solubilized in solubilizer buffer ( Tris, 50 mM, pH 7.8; NaCl, 300 mM; Triton X-100, 0.5 %) in presence of protease inhibitors (CompleteTM Roche Molecular Biochemicals, Indianapolis IN) and phosphatase inhibitors (NaF 100 mM; sodium pyrophosphate, 5 mM; sodium orthovanadate, 2 mM; EDTA, 5 mM).
RNA isolation, reverse transcription-PCR (RT-PCR) and Northern Analysis:
Total RNA was isolated using the Paris™ Kit (Ambion Inc Austin, TX ). RT-PCR was performed as previously reported in Suminami Y.et al. 13. Briefly, 10 g of total RNA was treated for 45 min with RNAse-free DNAse (Invitrogen) at 37°C to remove genomic DNA contamination, phenol/chloroform extracted and ethanol precipitated. RT reaction was performed using oligodT primers and the Suppererscript™ III RNase H- Kit (Invitrogen) for 60 min at 50°C. PCR reaction was performed using 10 ng of cDNA as follows: 5 min of denaturation at 94°C followed by 40 cycles of 1 min at 94°C, 1 min at 60°C, and 1 min at 72°C, followed by 7 min at 72°C, using the TaqGold DNA polymerase (2.5 U/reaction) (Roche Molecular Biochemicals, Indianapolis, IN). Amplified DNA fragments were visualized on 1.2% agarose gels. The primers used were as follows: 350S (sense) and 262797 (antisense) primers corresponded to amino acids 350-841 of the IL-2 receptor chain sequence 14. The expected length for the amplified product was 490 bp.
Northern blot and hybridization procedures were performed according to standard procedures. All cDNA probes were radiolabeled with Random Primers Synthesis Kit (Roche Diagnostics Gambh). The probe used in the northern study was obtained from RT-PCR for -chain, as described above. For Human Multiple tissue expression (MTE™) Array 2 (BD Biosciences), a nylon membrane containing normalized loadings of poly-A+RNA from 72 different human tissues and eight different control RNAs and DNAs were hybridized with the IL-2 receptor -chain probe. The membranes were independently hybridised three times with the radioactive probe, the tissues that consistently allowed the detection of the transcript were considered positive for chain expression. For Human tumor MTN Blot (BD biosciences), a premade Northern blot containing normalized loadings of poly-A+RNA (2g/ lane) from eight different human tissues was probed as described above, for IL-2 receptor -chain.
Binding analysis and scatchard plot:
2.0x106 A-498 Cells were incubated at 4°C for 12hrs in HEPES binding buffer with radiolabeled IL-2 (0.1 ng/ml, Perkin Elmer Boston Ma) in the absence or in presence of increasing concentrations of unlabeled ligand (6.6x10-9M, 1.0x10-8M, 1.5x10-8M, 2.3x10-8M, 3.6x10-8M, 5.0x10-8M, 6.3x10-8M, 8.3x10-8M, 8.3x10-7M). Counts detected in presence of the highest dose were considered a measure of non-specific binding (2%) and subtracted from the total counts. At the end of the incubation time the cells were washed 5 times with ice cold PBS( Gibco-Invitrogen) and resuspended in 5x Laemmli sample buffer. Cell bound activity was measured in a scintillation (gamma) counter. Specific binding was expressed as a percent of the binding capacity in absence of the cold ligand. For Scatchard plot analysis the data were analyzed with Sigmaplot 2000 graphing software.
D222A (Sgk1 dominant negative) continous cell line:
Hela cells were transfected with either pcDNA4TO D222ASgk1 or pcDNA4TO empty vector by Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. 48Hr after transfection cells were selected based on their the ability to grow in presence of Zeocin (400 g/ml). A total of 102 independent clones were isolated. 50 clones were pooled and the transgene expression was verified by western blotting with myc immunoglobulins, RT-PCR and immunofluorescence (data not shown).
Effect of PD89059 on cell proliferation
For the proliferative assay after Erk1/2 inhibition, the cell were plated at 5x104 and after 24 hours of serum starvation, exposed to 10M of ( Chalbiochem Darmstadt, Germany), or 10M of PD89059 plus 1.0x10-8M of IL-2, or 10% serum or left in basal condition and treated as described above. The data were expressed as a mean of the number of the cells counted in every condition .
Supplementary file 1
Panel A. Expression of IL-2Rβ chain in various human tumor tissues. Human Tumor MTN Blot (BD bioscience), containing normalized loadings (2μg) of poly A+ RNA per lane from eight different human tumor tissues were probed with 32P-labeled IL-2RcDNA obtained from RT-PCR performed in A-498 cells. Lanes 1-8, from left to right, contain RNA from the following tissues: breast, ovary, uterus, lung, kidney, stomach, colon, and rectum tumor tissue.
Panel B. Expression of IL-2Rβ chain in non-tumor human tissues. Human Multiple Tissue expression (MTETM) containing normalized loadings of poly A+RNA from 72 different human tissues and eight different control RNAs and DNAs were probed with 32P-labeled IL-2RcDNA obtained from RT-PCR performed in A-498 cells. The specific tissues positive for IL-2Rβ chain expression are described in the text. Only samples that were reproducibly positive in different reprobings were considered significant and encircled.
Supplementary file 2
Panel A. IL-2 prevents caspase 8 activation induced by doxorubicin, in serum starved cells.
A-498 cells were incubated for 24 hrs with albumin 0.1%, without ( lane 1) or with doxorubicin (doxo, 0.1μg/ml) ( lane 2) , or with doxorubicin (0.1μg/ml) after a 2 hr pre-treatment of IL-2 (1.0x10-8M) ( lane 3) . Cells were solubilized, proteins were separated by SDS-PAGE and probed for caspase 8. The upper band represents pro-caspase 8 and the lower band represents the cleaved form (activated) of caspase 8. Equal protein loading was confirmed by -tubulin blotting.
The graph shows a densitometric scanning of active caspase 8 in serum starved cells ; The intensity of the band corresponding to the active caspase is expressed as the percentage increase over the intensity of the band corresponding to the inactive caspase. Results are the average +/-S.E. of three independent experiments. AU = arbitrary units
Suppl. File 3
Detection of Sgk1 protein in A-498 cells. Sgk1 was detected in solubilized cell extracts by western blotting with a rabbit anti Sgk1 antibody ( upstate), in presence ( lane 2) and in absence ( lane 1) of the specific peptide, as indicated in the method section.
Suppl. file 4
Immunoprecipitation of IL-2-activated Sgk1.
A-498 cells were incubated for different times, as indicated, with or without IL-2 (1.0x10-8M). Solubilized cell extracts were immunoprecipitated with rabbit immunoglobulins specific for Sgk1 and separated by SDS-PAGE. Phoposhorylated Sgk1 was detected by immunoblotting with Goat immunoglobulins specific for phospho-Ser422 Sgk. Equal protein loading was confirmed by reprobing the filter with anti Sgk1 immunoglobulins (bottom).
Suppl. File 5
Panel A Interlukin protection from doxorucin induced caspase 8 activation in A498 cells in presence of Sgk1 specific si RNA.
The effect of IL-2 on doxorubicin induced caspase 8 activation was studied in A498 cells by means of scrambled or Sgk1 specific siRNA. An almost complete inhibition of Sgk1 expression was obtained in cells treated with Sgk1 specific siRNA (left ). Lanes 1 to 3 show the results obtained in cells expressing normal levels of Sgk1. In basal conditions ( lane 1) most of the caspase 8 in detected in the uncleaved, high molecular weight form. The addition of doxorubicin greatly decreases the uncleaved form leading to the appearance of highly degraded, low molecular weight peptides ( lane 2). IL-2 (lane 3) completely reverted the effect of doxorubicin to the pattern observed in absence of the drug . In absence of Sgk1 (Lanes 4 to 6) most of the caspase 8 is detected in the uncleaved high molecular weight form (Pro-Caspase) in absence of either doxorubicin or IL-2 ( lane 4). The addition of doxorubicin greatly decreases the uncleaved form leading to the appearance of highly degraded, low molecular wight peptides ( lane5). IL-2 (lane 6) was unable to revert the effect of doxorubicin to the pattern observed in absence of the drug.A densitometric scanning of three independent experiments as decribed in panel B is showed.
Panel B. Inhibition of the endogenous Sgk1 by the dominant negative mutant technology.
In A-498 cells transfected with a dominant negative mutant of Sgk1, IL-2 is unable to inhibit completely doxorubicin dependent caspase 8 activation. A-498 cells were transfected with a vector coding for a myc tagged dominant negative mutant ( D222A) of Sgk1 (upper gel). 48 hours after transfection cells were either serum starved for 12 hours (upper gel) or left in presence of 10% FBS ( bottom gel). Cells were then incubated for further 12 hrs without ( lane 1 ) or with doxorubicin (doxo, 0.1μg/ml) ( lane 2 ), or with doxorubicin (0.1μg/ml) after a 2 hr pre-treatment with IL-2 (1.0x10-8M) ( lane 3 ) . Cells were solubilized and the extracts were processed as in panels A and B. The expression of the transgene was verified by blotting with a myc antibody. Equal protein loading was confirmed by -tubulin blotting.