Sirt1 inhibitor-induced senescence-like growth arrest

H. Ota et al.

SUPPLEMENTARY INFORMATION

“Sirt1 inhibitor, Sirtinol, induces senescence-like growth arrest with attenuated Ras–MAPK signaling in human cancer cells”

Hidetaka Ota, Eriko Tokunaga, Kyungho Chang, Miyuki Hikasa, Katsuya Iijima, Masato Eto, Koichi Kozaki, Masahiro Akishita, Yasuyoshi Ouchi and Masao Kaneki

SA--gal staining

At 10 days after the addition of Sirtinol or Splitomicin to the culture media, the cells were washed twice with phosphate-buffered saline (PBS) and then fixed with PBS containing 2% formaldehyde and 0.2% glutaraldehyde for 10 min. The cells were then incubated at 37ºC for 10 hr with staining solution (40 mM citric acid sodium phosphate, pH 6.0, 1 mg/ml 5-bromo-4-chloro-3-isolyl--D-galactoside [X-gal, Fisher, Pittsburgh, PA], 5 mM potassium ferricyanide, 150 mM NaCl, 2 mM MgCl2). After being washed twice with PBS, the cells were incubated with 4’, 6-diamidino-2-phenlindole dihydrochloride (DAPI, 1 g/ml, DOJINDO, Tokyo, Japan) at 4C for 2 hr. SA--gal-positive cells were enumerated by counting over 400 cells in three independent fields.

BrdU incorporation assay

Cells were pulsed with 10 M 5-bromo-2’-deoxyuridine (BrdU, Molecular Probes, Eugene, OR) for 30 min. After the cells were washed twice with PBS and fixed with 70% ethanol at 37ºC for 30 min, DNA in the cells was denatured with 2N HCl at 37ºC for 1 h, followed by rinsing twice with PBS containing 0.5% Tween-20. The cells were then incubated with FITC-conjugated anti-BrdU antibody (Molecular Probes) at 37ºC for 30 min, and then stained with DAPI.

Immunoblot analysis

Cells were lysed on ice for 30 min in lysis buffer (50 mM HCl, pH 7.6, 150 mM NaCl, 1% NP-40, 0.1% sodium dodecyl sulfate (SDS), 1 mM dithiothreitol, 1 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride, 10 g/ml aprotinin, 10 g/ml leupeptin, 10 mM sodium fluoride). Equal amounts of protein were separated by SDS-polyacrylamide gel electrophoresis and then transferred to nitrocellulose filters. After being blocked with 5% dried milk in PBS containing 0.1% Tween 20, the filters were incubated with primary antibodies. After washing and incubation with horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse IgG (Amersham, Piscataway, NJ), the antigen-antibody complexes were visualized by chemiluminescence (ECL detection system; Perkin Elmer, Boston, MA).

Determination of activation status of Ras

At 10 days after the addition of Sirtinol, the cells were treated with or without EGF (50 ng/ml) for 20 min following overnight serum starvation. Cell lysates were incubated with recombinant protein of Ras binding domain of Raf-1 that was fused to glutathione-S-transferase. The absorbates to glutathione agarose were subjected to immunoblotting with anti-Pan-Ras antibody.

Colony formation assay

Cells were plated in 12-well plates at a density of 500 cells/well. At 24 h after inoculation of the cells, the culture media were replaced with media containing indicated concentrations of Sirtinol or Splitomicin. After exposure for 24 h, the cells were washed twice with PBS. Fresh medium was then added, and the cells were cultured for 14 days. The cells were stained with crystal violet (0.5% in 95% ethanol).

Supplementary Figure 1 Sirt1 inhibition results in induction of SA--gal in human normal fibroblasts. (a) WI-38 cells at population doubling level of 36 were treated with Sirtinol (100 M) for 24 hr. At 10 days after the addition of Sirtinol, the cells were subjected to SA--gal staining. (b) WI-38 cells were exposed to various concentrations of Sirtinol (30, 60, 100 M), Splitomicin (100, 300 M) or H2O2 (100, 150 M). At10 daysafter the addition of inhibitors or H2O2, the cells were subjected to SA--gal staining. (c, d) IMR-90 cells at population doubling level of 30 were treated with siRNA for Sirt1 or control siRNA. At 10 days after the addition of siRNA, SA--gal staining was performed. Protein expression of Sirt1 and -actin was evaluated by immunoblotting at 3 days after the addition of siRNA. **P<0.01 vs Control
Supplementary Figure 2 Sirtinol treatment did not alter p27 mRNA level in MCF-7 and H1299 cells. MCF-7 and H1299 cells were treated with Sirtinol (100 M) for 24 hr. At 3 and 10 days after the addition of Sirtinol, mRNA was isolated using ISOGEN mRNA purification kit (Nippon Gene, Toyama, Japan). cDNA probe for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was purchased from COSMO BIO (Tokyo, Japan). The mRNA levels of p27 and GAPDH were not altered by Sirtinol treatment in MCF-7 and H1299 cells at both 3 and 10 days after the addition of Sirtinol. In contrast, exposure to tamoxifen (1 M) for 24 hr and serum starvation for 24 hr increased p27 mRNA level in MCF-7 and H1299 cells, respectively.

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