Hct116 cells

Human embryonic kidney HEK293T cells (CRL-3216) and human colorectal adenocarcinoma SW480 (CCL-228) and HCT116 cells (CCL-247) were obtained from American Type Culture Collection. HEK293T cells were maintained in DMEM (Corning) supplemented with 10% FBS (Gibco) at 37°C in a humidified, 5% CO₂ incubator. HCT116 cells were maintained in McCoy’s 5a Modified Medium (Corning) supplemented with 10% FBS, while SW480 cells were maintained in Leibovitz’s L-15 Medium (Corning) supplemented with 10% FBS without CO₂. Cell transfection was conducted with VigoFect (Vigorous Biotechnology) or Lipofectamine 2000 (Invitrogen).
All plasmids used in this study were generated by subcloning corresponding cDNAs into HA-pcDNA3.1 (for mammalian cell expression), pET32M.3C, pETMBP.3C (for bacteria expression), or pCS107-HA (for in vitro transcription) vectors. The sequences encoding human Axin1 (NCBI RefSeq no. NM_181050.3), human APC (NM_000038.6), human GSK3β (NM_001146156.2), β-catenin (NM_001098209.2), and human CK1α (NM_001025105.3) were amplified using standard PCR procedures with cDNA from HEK293T cells and subcloned into HA-pcDNA3.1, pETMBP.3C, and pCS107-HA vectors. Drosophila melanogaster dAPC2 (NM_001347814.1) was amplified and subcloned into pETMBP.3C vectors. Plasmids encoding Axin1 deletions or mutations were generated by PCR using primers with appropriate nucleotide substitutions.

Human colon cancer HCT116 cells (ATCC) were grown in complete McCoy’s 5A medium (Corning, Corning, NY; 10050CV) supplemented with 10% fetal bovine serum (Gibco Laboratories, Gaithersburg, MD; A3160602). For generating stable short hairpin RNA (shRNA) knockdown cell lines, FOXO3-specific shRNA (shFOXO3) and control shRNA were cloned into the TET-ON all in one LT3GEPIR vector¹⁷ (link) by using the following nucleotide shRNA guide sequences: 5′-CATGTTCAATGGGAGCTTGGA-3′ (shFOXO3), 5′-TAGATAAGCATTATAATTCCTA-3′ (shCon). Sequence verified plasmids were transfected into HCT116 cells by using lipofectamine 3000 according to manufacturer’s instructions (Invitrogen, Carlsbad, CA; L3000001). Stable cell lines were generated from single cells by using puromycin selection (250 ng/mL; Sigma-Aldrich; P8833), and doxycycline-inducible (2 μg/mL; Sigma-Aldrich; D9891) knockdown of FOXO3 was validated by quantitative polymerase chain reaction (qPCR) and immunoblot.