Cell culture
The human hepatoma cell line Hep3B was obtained from the Cell Resource Center for Biomedical Research at the Institute of Development, Aging, and Cancer of Tohoku University (Sendai, Japan). Hep 3B cells were cultured in Dulbecco's Modified Eagle's Medium, high glucose (Wako Pure Chemicals, Osaka, Japan) supplemented with deactivated 10% fetal bovine serum FBS (Sigma-Aldrich, Missouri). Cells were cultured at 37°C with a CO₂ concentration of 5% in medium containing 10% deactivated fetal bovine serum (FBS; SIGMA-ALDRICH, Missouri) and 1% penicillin-streptomycin (SIGMA-ALDRICH, Missouri). Human lung cancer A549 (Resource No. RCB0098) cells were purchased from the RIKEN BioResource Center and cultured under the same conditions.
Treatment of cells with reagents
Sulforaphane (Cayman Chemicals, MI) was dissolved in 99.5% ethanol and added to Hep3B cells at a final concentration of 10 µM for 24 hours and to A549 cells at a final concentration of 30 µM for 8 hours. Akt inhibitor XI (Cayman Chemicals) was dissolved in DMSO and added to cells at a final concentration of 1 µM for 25 h in Hep3B cells and 9 h in A549 cells. The inhibitor was added one hour before the addition of SFN. A GSK3β inhibitor (SB216763, Tokyo Chemical Industry) was added at a final concentration of 2 µM for 25 hours in Hep3B cells and 9 hours in A549 cells.
Reverse transcriptase reaction and PCR
Total RNA was extracted from cells by Isogen (Nippon gene) according to the manufacturer’s instructions and cDNA was synthesized using Revert AidTM M-MuLV Reverse Transcriptase (New England Biolabs, Ipswich, MA). The reverse transcriptase reaction was performed at 25°C for 15 min, 42°C for 60 min, and 70°C for 10 min.
cDNA was then amplified with Go Taq polymerase (Madison, WS) and each primer set (Table 1).
Quantification of PCR products
PCR products were electrophoresed on a 2% agarose gel (Nippon gene, Tokyo, Japan) containing TAE buffer (400 mM Tris, 400 mM acetic acid, and 10 mM EDTA) plus ethidium bromide (Wako Pure Chemicals, Osaka, Japan). Bands were visualized using AE-6905H Image Save HR (ATTO) and quantified using ImageJ.
Immunofluorescence staining and immunoblotting
pcDNA/Flag-FOXO3 was prepared and transfected into Hep3B and A549 cells as previously described (18). Hep3B and A549 cells overexpressing Flag-FOXO were cultured and treated with 10 µM SFN and 1 µM AKT inhibitor XI for 25 hours. The primary antibody was an anti-DYKDDDDK tag antibody (Wako), and the secondary antibody, Alexa fluor 488-conjugated goat anti-mouse IgG (Abcam, Cambridge, MA) and DAPI(Chemical Dojin, Kumamoto, Japan) were used. The subcellular localization of proteins was then observed by confocal laser microscopy (Leica SP8, Leica MICROSYSTEMS). Immunoblotting was performed with anti-Cullin4A, DDB1(Santa Cruz Biotechnology, Dallas, TX), and phosphorylated FOXO3A (p-FOXO3A, Cell Signaling Technology , Danvers, MA) antibodies.
Experiments on C. elegans
The following strains were obtained from the Caenorhabditis Genetic Center (CGC, Minneapolis, MN) and used in the present study
1. wild-type strain N2 Bristol
2. vc1772 [skn-1(ok2315)/nT1(qIs51)]
3. cf1038 [daf-16(mu86)]
4. GR1310 [akt-1(mg144)]
The following strain was provided by the National BioResource Project (Tokyo, Japan).
1. wdr-23(tm1817)
The following strain was prepared in our laboratory.
1. skn-1(ok2315); ldIs7
C. elegans culture and reagent treatment
All C. elegans strains were maintained at 20°C on standard nematode growth medium (NGM) plates seeded with OP50 bacteria using standard techniques (18). SFN was dissolved in ethanol and mixed with OP50 to final concentrations of 25, 50, and 100 µM, and Akt inhibitor XI was mixed with OP50 to a final concentration of 4 µM and added to autoclaved NGM plates.
Lifespan assay
All experiments were performed on age-matched gilts synchronized with spawning. Lifespan assay experiments were conducted at 20°C. All nematodes were synchronized using a hypochlorite treatment, and late L4 larvae or young adult nematodes were transferred and defined as day 0 of the experiment. Nematodes were transferred to a new plate and scored every 2 days for survival observations. Nematodes that did not respond to repeated poking with a platinum wire were recorded as dead, and missing nematodes were not included in the death count and subtracted from the overall count.
Statistical Analysis
A t-test was used in statistical analyses of single comparisons between means. A one-way analysis of variance followed by Tukey’s post hoc test was performed for multiple comparisons. C. elegans longevity was plotted using a Kaplan-Meier survival analysis and compared between groups for significance using the Log-rank test. P <0.05 was considered to be significant (*p <0.05, **p <0.01, ***p <0.001).