Supplemental Data

Figure S1. DDR of mTOR-/- and mTOR+/+ Lin- cells in response to IR in vitro

Lin- cells were treated with or without IR (5 Gy) and then incubated for 7 h. Damaged DNA was detected by comet assay. Data are represented as mean ± SD. **P<0.01.

Figure S2. Chromosome breakage and radial formation in PD20 cells derived from human Fanconi anemia patient and deficient for FANCD2 (Hu-FANCD2-/-)

Hu-FANCD2-/- cells were cultured for two days in the presence or absence of MMC. Chromosomes were then subjected to cytogenetic analysis. A total of 50 cells from each sample were examined and scored. Arrowheads point to radial structures in the Hu-FANCD2-/- + MMC cells (a). Because too many breaks were observed in Hu-FANCD2-/- + MMC, the number of chromosome breaks per cell in these cells was artificially set to 10 (b). Data are represented as mean ± SD. **P<0.01.

Figure S3. FANCD2 expression in Raptor-/-, Raptor+/+, Rictor-/-, and Rictor+/+ Lin- cells

Expression of Raptor (a) and Rictor (b) in bone marrow cells was detected by Western blotting. FANCD2 expression was analyzed by quantitative RT-PCR. Data are represented as mean ± SD.

Figure S4. Effects of mTOR deficiency or inhibition on the canonical NF-κB target genes

(a) mTOR inhibition causes an increased NF-κB responsive luciferase reporter activity. NF-κB luciferase reporter construct was transfected into 293T cells. β-galactosidase construct was used as internal control. pp242 was added 24 h after transfection and the cells were cultured for another 24 h before the luciferase activity was assayed. The ratio of luciferase activities and β-galactosidase activities was shown. (b) mTOR deficiency causes an increased expression of NF-κB target genes. The expression of NF-κB target genes IL-1R2 and IGFR in mTOR-/- LSK cells was detected by quantitative real-time RT-PCR. Data are represented as mean ± SD. **P<0.01.

Figure S5. Inhibition of NF-κB rescues mTOR deficiency-induced DDR defects

Lin- cells were cultured for 24 h and then preincubated with or without JSH-23 for 30 min. The cells were then treated overnight with or without MMC. γH2AX foci formation was visualized by immunofluorescence (a, b). The percentage of positive cells (≥ 6 γH2AX foci) was assessed from 30-50 nuclei and quantified (c).

Figure S6. Complex formation of mTOR with IKK and of IKK with TAK1

(a) Hu-FANCD2+/+ cells were treated for 12 h with or without pp242 and subjected to immunoprecipitation by using anti-mTOR antibody or control IgG. mTOR and IKKα were then detected by Western blotting. (b)

Hu-FANCD2+/+ cells were treated for 12 h with or without pp242 and subjected to immunoprecipitation by using anti-IKKα antibody or control IgG. IKKα and TAK1 were then detected by Western blotting.

Supplemental Materials and Methods

Mice. Conditional gene-targeted Raptorloxp/loxp or Rictorloxp/loxp mice were generated as described previously. 1, 2 The flox allele contains loxP sites flanking exon 6 of Raptor gene or exon 3 of Rictor gene. To delete Raptor or Rictor in vivo in hematopoietic stem cells, Raptorloxp/loxp; Mx-Cre+ or Rictorloxp/loxp;Mx-Cre+ mice were generated by breeding Raptorloxp/loxp or Rictorloxp/loxp mice with Mx-Cre+ transgenic mice carrying a bacteriophage Cre recombinase driven by an interferon-α-inducible Mx1 promoter. The expression of Cre was induced by 6 to 8 i.p. injections of 10 μg/g of body weight pIpC (Amersham Pharmacia Biotech, Piscataway, NJ) into the Mx-Cre+ mice at 2-day intervals

Western blot

Whole-cell lysates were prepared and separated by 10% SDS-polyacrylamide gel electrophoresis. The expression of Raptor and Rictor was probed by using corresponding antibodies (Cell Signaling Technology).

Quantitative real-time RT-PCR. Total RNA was isolated by using RNeasy Micro Kit (Qiagen) and recommended protocol. Reverse transcription was performed with random hexamers and Superscript II RT (Invitrogen) and was carried out at 42 °C for 60 min and stopped at 95 °C for 5 min. Real-time quantitative PCR was carried out in an ABI Prism 7900 Sequence Detector by using SYBR Green PCR Master Mix reagent (Applied Biosystems). Primer sequences used in this study are: mouse IL-1R2, forward 5’-ctggaaggtgaacctgtggt-3’, reverse 5’-catttgctcacagtgggatg-3’; mouse IGFR, forward 5’-ttaccgtttgggagaactgg-3’, reverse 5’-cttgacccacaacctgacct-3’.

NF-κB luciferase reporter assay. The NF-κB luciferase reporter construct (Stratagene) was transiently co-expressed with cDNA encoding β-galactosidase. Transient transfection of the reporter plasmids was carried out by using FuGENE 6 Transfection Reagent (Roche Applied Science) according to the manufacturer's protocols. Twenty-four hours after transfection, pp242 was added to the cell culture. The cells were incubated for another 24 h. Analysis of luciferase and β-galactosidase activities of the transfected cells was performed by using a luciferase assay kit (Promega). Transfection efficiencies were routinely corrected by obtaining the ratio of the luciferase and the β-galactosidase activities observed in the same sample.

Immunoprecipitation. Cells were lysed on ice for 20 min in 1 mL of lysis buffer (40 mM Hepes at pH 7.5, 120 mM NaCl, 1 mM EDTA, 10 mM pyrophosphate, 10 mM glycerophosphate, 50 mM NaF, 0.5 mM orthovanadate, EDTA-free protease inhibitors [Roche]) containing 0.3% CHAPS. After centrifugation at 12,000g for 10 min, mTOR antibody, IKKα antibody or control IgG was added to the cell lysates and incubated with rotation for overnight. 25 μL of protein A/G-agarose were added and the incubation continued for 1 h. Immunoprecipitates captured with protein A/G-agarose were washed three times with the lysis Buffer and two times by wash buffer A (50 mM Hepes at pH 7.5, 150 mM NaCl), and boiled in 5× SDS sample buffer prior to electrophoresis and immunoblotting.

Statistical analysis. All experimental data were analyzed and compared for statistically significant differences by two-tailed Student's t test. Data are presented as the averaged values ± standard deviations (SD) where applicable. The following values were considered significant: *, P < 0.05, and **, P < 0.01.

Supplemental References

1.  Sengupta S, Peterson TR, Laplante M, Oh S, Sabatini DM. mTORC1 controls fasting-induced ketogenesis and its modulation by ageing. Nature 2010; 468:1100-1104.

2.  Shiota C, Woo JT, Lindner J, Shelton KD, Magnuson MA. Multiallelic disruption of the rictor gene in mice reveals that mTOR complex 2 is essential for fetal growth and viability. Dev Cell 2006;

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