After this interval, the culture media containing MTT were discarded and DMSO was added to each well, dissolving the precipitate. The optical densities were measured at 560nm spectral wavelength using microtitre plate reader (Synergy HT Multi-Mode Microplate Reader; Bio-Tek Instruments Inc., Winooski, VT, USA). Transfection and luciferase reporter assay Transient technical support transfection of HepG2 human hepatocytes was performed using the TransFectin reagent (Bio-Rad, Hercules, CA, USA). Constructs used were the FHRE-Luciferase reporter (Addgene plasmid 1789 kindly provided by M Greenberg’s lab) (Tran et al, 2002) and the FoxO3a expression construct (Addgene plasmid 8355 kindly provided by A Brunet’s lab) (Brunet et al, 1999). Inducible activation of FoxO3a was performed through transfection of the HA-FoxO3a-WT-ER plasmid.

The HA-FoxO3a-WT-ER fusion protein is constitutively expressed but is inhibited unless exposed to a modified ligand for the oestrogen receptor (ER), 4-hydroxy-tamoxifen (4-OHT). HepG2 cells were transfected using the TransFectin reagent (Bio-Rad, Munich, Germany) with 1��g of HA-FoxO3a-WT-ER plasmid (Tran et al, 2002). Activation of the accumulated FoxO3a protein was induced by treatment with the ER ligand 4-OHT 1h before melatonin treatment. The luciferase reporter activity was measured using a commercially available luciferase assay system (Promega). Transfection efficiency was normalised by ��-galactosidase activity. Western blot analysis After treatments, cultured cells were washed two times with ice-cold PBS and lysed by adding ice-cold RIPA buffer containing 50m Tris-HCl (pH 7.

4), 150m NaCl, 2m EDTA, 0.1% Triton X-100, 10% sodium deoxycholate, 10% SDS, 1m NaF and protease cocktail inhibitor (Roche, Basel, Switzerland), and scraped off the plate. The extracts were transferred to a microfuge tube and centrifuged for 10min at 15000g. Equal amounts of the supernatant protein (20��g) were separately subjected to SDS�CPAGE and transferred to a PVDF membrane (Bio-Rad). Primary Abs were diluted in blocking solution and incubated overnight at 4��C with polyclonal Ab to Bim (rabbit, 1:1000 dilution; eBioscience, San Diego, CA, USA), phospho-FoxO3a Thr32 and FoxO3a Ser253(rabbit, 1:1000 dilution; Cell Signalling Technology, Beverly, MA, USA) and FoxO3a (rabbit, 1:1000 dilution; Abcam, Cambridge, UK).

Equal loading of protein was demonstrated by probing the membranes with a rabbit anti-��-actin polyclonal Ab (1:2000 dilution; Sigma), anti-lamin B1 (H-90) (1:2000 dilution; Santa Cruz AV-951 Biotechnology, Santa Cruz, CA, USA) or anti-tubulin (Sigma-Aldrich). After washing with PBST, membranes where incubated with phosphatase-conjugated anti-rabbit secondary Ab from Sigma-Aldrich diluted in blocking solution and incubated for 1h at room temperature.