Design of qRT-PCR probes to long and short FOXO3 isoforms
The sequences of FOXO3 transcripts corresponding to long and short FOXO3 isoforms were accessed from the ENSEMBL genome browser (https://www.ensembl.org), and assays specific to the two full length isoforms (FOXO3-FL; ENST00000343882.10 and ENST00000406360.2) or the truncated FOXO3 isoform (FOXO3-TR; ENST00000540898.1) were designed. The FOXO3-FL assay was targeted over the exon 2/exon 3 boundary shared by both full length isoforms and thus did not differentiate between them. The FOXO3-TR assay was targeted over the exon 1a/exon 3 boundary and was specific to ENST00000540898.1 (Fig. 1). Probes and primers were as follows: FOXO3-FL; forward primer — GGATAAGGGCGACAGCAACA, a reverse primer —GAATCGACTATGCAGTGACAGGTT and probe — FAM-CCGGATGGAGTTCTTC-MBG. FOXO3-TR; forward primer — GCCCTGGAACCTTTTGGCTTAA, reverse primer — CGACTATGCAGTGACAGGTTGT and probe FAM–CTCGGAAAACAAACTCC-MGB. Assays were purchased from Thermo Fisher (Waltham, USA).
Validation of FOXO3 isoform-specific assays
The performance of FOXO3-FL and FOXO3-TR assays was assessed using standard curve analysis. One hundred nanograms of skeletal muscle total RNA was reverse transcribed for each sample in a total volume of 20µl using the Evoscript cDNA synthesis kit (Roche, Burgess Hill, UK) according to the manufacturer’s instructions. cDNA then underwent seven 1:2 serial dilutions to establish accuracy, sensitivity and linear range. Reactions contained 2.5 µl TaqMan Universal Mastermix II (Thermo Fisher, Waltham, USA) 0.9µM each primer, 0.25µM probe (Thermo Fisher, Waltham, USA), 1.25µl H2O and 1µl cDNA in a total volume of 5µl. Cycling conditions were 95 OC for 10 min prior to cycling, then 95 OC for 15 s and 60 OC for a minute for 40 cycles.
Characterisation of the expression profile of FOXO3-FL and FOXO3-TR in human tissues
Samples from different human tissues (skeletal muscle, pancreas, lung, pons, ovary, oesophagus, adipose, bladder, thyroid, breast, testes, adrenal gland, small intestine, thymus, trachea, cervix, prostate, medulla oblongata, colon, stomach, postcentral gyrus, kidney, hippocampus, spleen, salivary gland, parietal lobe, liver, cerebellum, heart, cerebral cortex, placenta, temporal lobe, uterus, total brain, pituitary, bone marrow, pooled islets and blood) were obtained from commercially-sourced, tissue-specific RNA panels (Becton, Dickinson & Co., Franklin Lakes, NJ, USA: BioChain Institute, Newark, CA, USA: Ambion®, Austin, TX, USA). One hundred nanograms of total RNA was reverse transcribed for each sample in a total volume of 20µl using the Evoscript cDNA synthesis kit (Roche, Burgess Hill, UK) according to the manufacturer’s instructions. PCR reactions contained 2.5µl TaqMan Universal Mastermix (no AMPerase) (Applied Biosystems, Foster City, USA), 0.9µM each primer, 0.25µM probe, 1.25µl H2O and 1µl cDNA reverse transcribed as above in a total volume of 5µl. PCR conditions were a single cycle of 95ºC for 10 min followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The amount of FOXO3-FL and FOXO3-TR isoforms in each tissue sample was then quantified by isoform-specific qRT-PCR by the Comparative Ct method relative to the geometric mean of Beta 2 Microglobulin (B2M), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and beta glucoronidase (GUSB) endogenous control gene expression, with adjustment for assay efficiencies. Expression levels were then normalised to the average level of FOXO3-FL isoforms over the entire tissue panel.
Bioinformatic analysis of potential FOXO3 regulatory regions
To determine for rs2802292, rs2764264 and rs13217795 to interfere with splicing or expression of the FOXO3 gene, we firstly determined all the proxy SNPS that were in complete linkage disequilibrium (D’ = > 0.95, r2 > 0.80) with these index SNPs using SNPsnap (https://data.broadinstitute.org/mpg/snpsnap/about.html). Index SNPs and proxies were then mapped to the known regulatory regions of the FOXO3 gene with reference to the UCSC genome browser (https://www.genome.ucsc.edu/). SNPs were designated for follow-up if they were located within a predicted promoter element or within 50 bp of a splice site.
Profiling of FOXO3 isoforms in peripheral blood samples
We next profiled FOXO3 isoform levels in RNA extracted from 51 previously genotyped peripheral blood samples from the Exeter 10,000/Peninsula Research Bank study (https://exetercrfnihr.org/about/exeter-10000-prb/) selected on the basis of rs13217795 genotype. This collection is a cross sectional population study consisting of samples collected from volunteer individuals living in the South West of England and recruited since 2010. Whole blood samples were collected in 2011/2012 using the PAXgene system  and extracted using the PAXgene Blood RNA kit (Qiagen, Paisley, UK). Access to these anonymised samples was approved by the ethically approved Peninsula Research Bank Steering Committee (14/SW/1089). Our sample set consisted of 18 individuals homozygous for the major allele of rs13217795 (TT), 17 samples heterozygous for rs13217795 (CT) and 17 samples homozygous for the longevity allele of rs13217795 (CC). Participant characteristics are given in Table 1. Levels of FOXO3-FL and FOXO3-TR isoforms were then quantified relative to the geometric mean of the B2M and GAPDH genes by qRT-PCR as described above, with adjustment for assay efficiencies. Data were then normalised to the mean level of the full-length isoforms in the rs13217795 heterozygous samples. Statistical significance was then determined by ANOVA comparing age, sex and body mass index (BMI) covariates by genotype. Significant covariates were taken into account within a univariate linear model using the IBM SPSS Statistics 26 program (IBM SPSS version 26 release 22.214.171.124, Armonk, NY).
Characterisation of the expression profile of FOXO3-FL and FOXO3-TR in human skeletal muscle
In total, 64 human skeletal muscle samples were obtained from previously published studies of human muscle metabolism [6, 18, 21, 29]. Four samples were omitted due to unsuccessful genotyping. All volunteers abstained from alcohol and strenuous exercise for at least 48 h prior to the sample obtained. Samples were obtained from the vastus lateralis muscle and were dissected free of visible connective tissue, fat and blood immediately prior to snap freezing. Patient characteristics are given in Table 1. RNA was extracted from 20 mg homogenised muscle sample using TRIzol reagent (Thermo Fisher, Waltham, USA). RNA concentration was quantified by ND-2000 Nanodrop Spectrophotometer (NanoDrop,Thermo Fisher, Waltham City, USA). The genotype of each sample was determined by qPCR from the presence of heterogeneous RNA (hnRNA) in the RNA samples using 2.5 µl TaqMan Universal Mastermix II (Thermo Fisher, Waltham City, USA), 0.9µM of each primer and 0.2µM of each probe (genotyping assay ID = C_9174543_10) and 1 µl RNA in a total volume of 5 µl. Cycling conditions were 60 °C for 30 s, followed by 95 °C for 10 min prior to cycling. Samples were then cycled between 95 °C for 15 s and 60 °C for a minute for 40 cycles. Following this, samples were held at 60 °C for 30 s. FOXO3 isoform levels were quantified by qRT-PCR as described above. Levels of FOXO3-FL and FOXO3-TR isoforms were then quantified relative to the geometric mean of the B2M and GAPDH genes by qRT-PCR as described above, with adjustment for assay efficiencies. GUSB was omitted from this analysis as it proved unstable baseline in our sample set. Data were then normalised to the mean level of the full length isoforms in the rs13217795 heterozygous samples. Statistical significance was then determined by ANOVA comparing age, sex and body mass index (BMI) covariates by genotype. Significant covariates were taken into account within a univariate linear model using the IBM SPSS Statistics 26 program (IBM SPSS version 26 release 126.96.36.199, Armonk, NY).
Assessment of FOXO3-FL isoform expression by age in human peripheral blood
To determine whether FOXO3 isoform expression alters with age, we carried out an assessment of the association between age and FOXO3-FL isoform expression exclusively in the 16 individuals homozygous for the major (T/T) allele, to negate the effect of genotype by linear regression analysis. We were unfortunately unable to assess the effect of age on the truncated FOXO3-TR isoform since this isoform was only expressed in skeletal muscle, and the age range of the donors of our muscle samples was very limited (18 to 31 years).