H5784

CASIN (Xcessbio #M60040) was prepared freshly before injection. To this, it was dissolved in DMSO to a concentration of 100 mM and diluted in cyclodextrin solution (Sigma #H5784). IP injections of 25 mg/kg were done on 4 consecutive days in the morning starting at an age of 77–86 weeks. Control mice were injected with equal volume of the solvents. Mice were sacrificed 8–9 weeks after treatment, and blood was collected by heart puncture. The analysis of DNA methylation levels was analyzed at three age‐associated CG dinucleotides (CpGs) as described previously (Brown et al., 2020; Han et al., 2018). Briefly, genomic DNA was isolated from blood samples, bisulfite converted, and DNA methylation was analyzed within the three genes (Prima1, Hsf4, and Kcns1) by pyrosequencing. The DNA methylation results at these sites were integrated into a multivariable model for epigenetic age predictions in B6 mice, which clearly correlate with the chronological age (Han et al., 2018). Chronological and biological age of CASIN‐treated and control mice was compared using an unpaired t test. Since the data were not normally distributed, all data were potentiated to obtain normal distribution before the statistical analysis was performed.

Remodelin toxicity was assessed by the Crown Biosciences on 12 6-week-old C57BL/6N female mice, with a BW of 20 g on average (see Table S2. Animal supplier: Shanghai Laboratory Animal Center SLAC, Shanghai, China). At WTSI, Remodelin and Remodelin Fluor were dissolved in a solution of 20% DMSO, 65% (45% 2-Hydroxypropyl-b-cyclodextrin solution, H5784 Sigma Aldrich) and 15% Tween 80 (P8074 Sigma Aldrich). The vehicle-treated animals were given this solution alone, without Remodelin. Remodelin and Remodelin Fluor were administered daily by oral gavage at 100 mg per kg per day and 50 mg per kg per day respectively (defined as non-toxic doses in toxicity studies), from day 21 and until culled. Animals have been killed by CO₂ inhalation followed by cervical dislocation.