β naphthoflavone

All experiments were performed following the UK Home Office guidelines. All mice were maintained under non-barrier conditions and given a standard diet and water ad libitum. The following mouse strains were used: VilCre^ER (ref. ^{25 (link)}), Lgr5Cre^ER (ref. ^{26 (link)}), AhCre^ER (ref. ^{27 (link)}), R26R-LSL-tdTomato (tdTom^fl) (ref. ^{28 (link)}), Apc^fl (ref. ^{29 (link)}), Catnb^lox(ex3) (ref. ^{30 (link)}), Braf^{V600 E} (ref. ^{31 (link)}), PTEN^{f l} (ref. ^{32 (link)}). The Lgr5Cre^ERApc^fl/fl and VilCre^ERApc^fl/+ mice were on a C57/B6 background (backcrossed ≥10 generations). The Porcupine inhibitor LGK974 (also referred to as WNT974) was administered in a concentration of 5 mg/kg BID (oral) in a vehicle of 0.5% Tween-80/0.5% methylcellulose. AhCre^ER mice were induced with 1 mg β-naphthoflavone (Sigma) and 0.15 mg tamoxifen (Sigma) IP. VilCre^ER and Lgr5Cre^ER mice were induced with tamoxifen (Sigma) IP at the concentrations indicated throughout the manuscript.

Acenaphthenol, benzyloxyresorufin, bicinchoninic acid (BCA) assay kit, cytochrome c, NADH, NADPH, NADP⁺, 7-ethoxyresorufine, menadione, 7-methoxyresorufine, β-naphthoflavone, p-nitrophenyl sulfate, resorufin, R-sulforaphane, 3′-phosphoadenosin-5′-phosphate, and UDP-glucuronic acid were purchased from Sigma-Aldrich (Prague, Czech Republic). Midazolam was obtained from Toronto Research Chemicals (North York, ON, Canada). All other chemicals used were of HPLC or analytical grade. For real-time Polymerase Chain Reaction (PCR) analyses, ProtoScript^® II Reverse Transcriptase was purchased from NEB (Ipswich, UK), TriReagent from Biotech (Prague, Czech Republic), and the qPCRCore kit for SYBR Green I from Eurogentec (Seraing, Belgium).

Animals were maintained on an outbred background, housed in a standard facility and all experimental procedures were performed in accordance with institutional animal care and ARRIVE guidelines in compliance with UK Home Office regulations. In brief, mice were maintained in conventional open top cages on dust free bedding (IPS Ltd) under a 12 hr light cycle, with RM3(E) diet (Special Diet Services UK) provided for nutritional support. To enrich the environment, sunflower seeds (at weaning only, LBS Ltd), nestlets (IPS Ltd), disposable envirotubes (IPS Ltd) and small chewsticks (Labdiet–IPS Ltd) were provided. Mice carrying the targeted Lect2 allele were kindly supplied by Dr Satoshi Yamagoe [5 (link)]. Experimental mice were genotyped as previously described for the targeted Lect2 allele [5 (link)], Apc allele [10 (link)], the Apc^Min/+ allele [39 (link)], the Rosa26R allele [40 (link)] and the AhCre transgene [11 (link)]. Cre activity was induced in control and experimental mice by 3 consecutive intraperitoneal (i.p.) injections of 80 mg/kg β-naphthoflavone (Sigma, UK) in 24 h. Mixed sex control (litter mates) and experimental mice were used for timepoint (N > 4) or survival (N > 15) experiments. Prior to proliferation analysis selected animals were injected with 100 μg/kg Bromo-deoxyuridine (Sigma, UK) and culled at indicated time points after labelling.

Pericarpium Citri Reticulatae was purchased from a local herbal market. It was authenticated by Professor Xin-feng Gao. A voucher specimen was deposited in Herbaruim of Chengdu Institute of Biology, No. CIBI0064257.
Nobiletin and Tangeretin were purchased from Chengdu Must Bio-technology Co., LTD (China) and indentified in our laboratory for qualitative and quantitative analysis. β-naphthoflavone, polydiallyldimethylammonium chloride (PDDA), β-nicotinamide adenine dinucleotide phosphate hydrate (NADP), glucose-6-phosphate, yeast glucose-6-phosphate dehydrogenase, 4-nitrophenol (PNP) and 4-nitrocatechol (PNC) were purchased from Sigma (MO, USA). HPLC grade acetonitrile was purchased from Fisher Scientific (Fisher, Fair Lawn, USA). Deionized water was purified by a Milli-Q water system (Millipore Corp., Bedford, MA, USA). Tetraethyl orthosilicate (TEOS) was purchased from TCI (Tokyo, Japan). Other chemicals and solvents were of analytical reagent grade and were obtained from Chengdu Chemical Factory (Chengdu, China).