a, b, Ghr and Igf1r gene expression changes measured by quantitative real-time PCR (qRT–PCR) in liver samples of 11-week old wild-type and Ercc1∆/− mice with restricted diets (n = 5). Gene-specific real-time PCR primers are described in Methods. c, MicroRNA expression profile comparison of wild-type and Ercc1∆/− mouse liver tissue under ad libitum and diet-restricted conditions. Shown are 188 significantly regulated miRNAs (FRD ≤ 5%) between groups. Five of the most significantly changed microRNAs are zoomed in. miR-34a, a downstream target of p53 that is involved in cell cycle regulation and apoptosis, is induced by DNA damage42,43. It showed differential expression between liver homogenates of 11-week-old wild-type and Ercc1∆/− mice. It was downregulated by dietary restriction in the liver of wild-type mice (1.62 fold, P = 0.02), but strongly upregulated in the liver of ALErcc1 mice compared to ALWT mice (4.7-fold, P = 0.0001) and seems suppressed in DRErcc1 expression profiles. These changes were confirmed by qPCR (data not shown). d, Heat map of key antioxidant defence genes in liver and brain of wild-type and Ercc1∆/− mice. Fold changes were calculated for DRWT, ALErcc1, and DRErcc1mice against ALWT mice, using microarray expression profiles of liver tissue at 11 weeks of age (n = 5) or qRT–PCR for cerebellum tissue at 16 weeks of age (n = 4). Dietary restriction induced an antioxidant response in liver, which is less pronounced in brain specimens, consistent with earlier findings44. This is likely to be due to the high endogenous antioxidant defence levels in the nervous system. The difference in antioxidant response between liver and brain by genotype conforms to previous results6. Interestingly, the Purkinje neuron marker calbindin is clearly reduced in cerebella of ALErcc1 mice but is less reduced in DRErcc1 mice, confirming the strong reduction in DNA-damage-induced Purkinje cell loss induced by dietary restriction. Blue, decreased expression; red, increased expression. Hierarchical clustering on liver and cerebellum genes was performed using a Pearson correlation. e, Dietary restriction reduces the p16-RB branch of senescence and the senescence-associated secretary phenotype (SASP) as assessed by next-generation sequencing expression analysis of the liver RNA of Ercc1∆/− mice. To assess the p16-RB branch of the senescence phenotype45, we followed a next-generation sequencing approach as previously described36 using 16-week-old liver tissue from ALWT, DRWT, ALErcc1 and DRErcc1 mice (n = 1). By sequencing >150M sequence reads per sample, we detected the p16-ink4a (Cdkn2a) transcript at sufficient levels. p16-ink4 (Cdkn2a) is considered a key marker for cellular senescence, but is difficult to quantitatively analyse using other methods owing to the high ratio of normal cells to senescent cells. Data sets were normalized by calculating reads per kilobase million (RPKM). Subsequently, z-scores were calculated and plotted in a heat map. Red, increased expression; blue, decreased expression. In ALErcc1- liver RNA, p16-ink4a (Cdkn2a) is upregulated compared to levels in ALWT animals, but downregulated after dietary restriction. This indicates that Ercc1∆/− mice have increased cellular senescence that is reduced upon dietary restriction. Second, we monitored the transcriptionally induced SASP as described previously46. Many, if not all, SASP factors are not exclusively specific for cellular senescence. To reduce the probability that observed SASP factor expression changes are contributed to by other cells, we selected only those SASP factors that have an absolute expression (RPKM) in the same range as p16-ink4a in across these data sets, since these are most likely to be the result of cellular senescence. The figure shows that most SASP factors such as IL-6, the most prominent SASP cytokine are downregulated after dietary restriction. This supports the idea that cellular senescence and associated SASP are increased in ALErcc1 liver and are reduced by dietary restriction. Hierarchical clustering was performed using a Pearson correlation. f, Suppression of long genes in normal ageing of rat liver. A relative-frequency plot of the gene length of DEGs in liver tissue from 24-month old rat versus that seen in 6-month old rat. Upregulated genes, red; downregulated genes, green. The DEGs from rat liver were selected using a fold-change cut-off of 1.5 and an FDR <0.05. The data set is publicly available in the NCBI Gene Expression Omnibus under accession number GSE66715.