Fig 2: Transcriptional response to cysteine deficiency.a, GO enrichment categories from liver comparing Het and KO mice on No Cys diet. b, Gdf15 RFPKM values from liver across all groups. c, Fgf21 RFPKM values from liver across all groups. d, GO enrichment categories from muscle comparing Het and KO mice on No Cys diet. e-g, Muscle bulk RNA-Seq data represented as heatmap for genes in oxidative stress, cellular metabolic process, branched chain amino acid metabolism, NAD, and pantetheinase that are upregulated (e), for genes in extracellular organization and animal organ development that are specifically downregulated (f), and for genes in the ISR that appear minimally affected (g) in No Cys KO mice at Day 2. h, GO enrichment categories from epididymal fat pad comparing Het and KO mice on No Cys diet. i, Ucp1 RFPKM values from epididymal fat pad. All data are at Day 2 based on mice shown in the RNA-seq schematic in Figure 3a. For GO enrichment table, Red indicates upregulated in KO and blue indicates downregulated.

Fig 3: General EAA deficiency coupled with a deficiency in GSH partially phenocopies cysteine deficiency.a, GSH levels in liver and muscle of CR male Het and KO at Day 2 on a No Cys diet (n=4, n≥3). b, Representative IHC staining (of n=4) of NRF2 and NQO1 in liver of Het and KO mice on CR No Cys diet at Day 3. c, P-eIF2α/ eIF2α ratio in Het and KO mice on CR No Cys diet at Day 2 (n≥5). d, GDF15 and e, FGF21 serum level at Day 2 of CR No Cys diet (n≥7 for GDF15, n≥4 for FGF21). f, Acetyl-CoA carboxylase 1 (ACC1) protein levels normalized to β-tubulin in Het and KO No Cys at Day 2 on CR No Cys diet (n≥4).g, FFA and h, TG and serum levels at Day 2 of CR No Cys diet (n≥5 for FFA and TG)i, Weight measurements of Cse Het and KO mice on different diets (n>=4 in each group). j,k, Bulk liver RNA-seq data for multiple groups represented as heatmap for genes in Integrated Stress response and oxidative stress responses (g), and those related to cholesterol and lipid metabolism (h). l-o, P-eIF2α/ eIF2α ratio in liver (l), liver GSH (m), ACC1 protein levels normalized to β-actin (n), and serum GDF15 (o) in calorie-restricted KO No Cys compared to KO No Trp + BSO mice at Day 2 (n≥4 for all). p, Summary of effects in KO mice by No Trp, No Cys and No Trp+BSO diets.

Fig 4: Effect of Cysteine and methionine dual restriction on wild type C57BL/6 mice.a, Weight loss curves of calorie restricted (2.1 g/day) male mice on Ctrl, No Met or No Met No Cys diet (n=4). b, Serum GDF15 levels across all three conditions on Day 3 or c, Day 7. Serum FGF21 levels across all three conditions on d, Day 3 or e, Day7 (n=4). f, Weight loss curves of male Gdf15 KO or WT mice on No Met No Cys diet (n≥3) g, Weight after one day of No Met No Cys diet in Gdf15 KO or WT mice h, Weight loss curves of female Fgf21 KO or WT mice on No Met No Cys diet (n≥4) i, Weight after one day of No Met No Cys diet in Fgf21 KO or WT mice.

Fig 5: Effect of cysteine restriction on tissue GSH, serum GDF15 and FGF21 levels.a-e, GSH levels in liver (a), muscle (b) and subcutaneous fat pad (c) GDF15 (d) and FGF21 (e) levels in serum from Cse Het and KO mice on Day 2 of CR diets without Cys or Trp or Ctrl diet.