SUPPLEMENTARY INFORMATION

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Ronald Atkinson
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1 DOI: /ncb3461 In the format provided by the authors and unedited. Supplementary Figure 1 (associated to Figure 1). Cpeb4 gene-targeted mice develop liver steatosis. a, Immunoblot displaying CPEB4 and α-tubulin protein levels in Cpeb4 +/+, Cpeb4 +/- and Cpeb4 -/- liver extracts. Unprocessed scans of original blots are shown in Supplementary Fig.7g. b, CPEB4 immunohistochemistry in WT and Cpeb4 KO liver sections. Scale bar, 100 μm. c, Cpeb4 mrna expression, normalized to TBP transcript levels, in livers from WT (n=8) and Cpeb4 KO (n=8) mice. Two-sided Student's t test, ***P= d, Changes in body weight of WT (n=34) and Cpeb4 KO (n=32) mice fed standard diet. Two-way ANOVA, P= e, Fed and overnight-fasted plasma glucose levels of mice fed HFD. WT-Fed, n=8; WT- Fasted, n=16; Cpeb4 KO -Fed, n=8; Cpeb4 KO -Fasted, n=14 mice. Two-way ANOVA, *P= f, Changes in body weight of WT (n=40) and Cpeb4 KO (n=28) mice fed HFD. Two-way ANOVA, *P= g, Body weight of WT (n=16) and Cpeb4 KO (n=10) mice aged for 80 weeks. Two-sided Student's t test, *P= h-j, 24-hour time course of RER (h), EE (i) and locomotor activity (j) of mice fed standard diet; WT, n=12; Cpeb4 KO, n=12 mice. Two-way ANOVA. k, Food intake (g/day) of WT (n=8) and Cpeb4 KO (n=8) mice on HFD during 4 consecutive days. Two-way ANOVA, P= l, Water intake in a 24-h period of WT (n=12) and Cpeb4 KO (n=12) mice fed standard diet. Two-sided Student's t test, P= m, Plasma glucose levels of WT and Cpeb4 KO fed, 6-h, and 24-h-fasted mice. WT-Fed, n=20; WT-6hFasted, n=22; WT-24hFasted, n=22; Cpeb4 KO -Fed, n=20; Cpeb4 KO - 6hFasted, n=20 mice; Cpeb4 KO -24hFasted, n=20 mice. Two-way ANOVA, P= n-o, Fed and overnight fasted plasma insulin levels (n) and free fatty acid (FFA) plasma levels (o). Panel n: WT-Fed, n=16; WT-Fasted, n=18; Cpeb4 KO -Fed, n=18; Cpeb4 KO -Fasted, n=16 mice. Panel o: WT- Fed, n=16; WT-Fasted, n=18; Cpeb4 KO -Fed, n=18; Cpeb4 KO -Fasted, n=16 mice. Two-way ANOVA, P= (n), P= (o). p-q, Glucose levels (p) and insulin levels (q) during glucose tolerance test in WT and Cpeb4 KO mice. Panel p: WT, n=18; Cpeb4 KO, n=18 mice. Panel q: WT, n=12; Cpeb4 KO, n=12 mice. Two-way ANOVA, P= (p), P= (q). r, Glucagon tolerance test after 6h fasting in WT (n=18) and Cpeb4 KO (n=18) mice. Two-way ANOVA, P= s, Glucose produced by primary hepatocytes in culture after treatment for 4h with vehicle (dimethyl sulfoxide, DMSO), with a combination of 10 μm forskolin, 20 mm lactate, and 2 mm pyruvate (FSK) or with a combination of 300 μm dibutyryl-camp and 100 nm dexamethasone (camp); n=12 primary hepatocyte cell lines from independent animals. Two-way ANOVA, P= For c-s, data are mean±s.e.m. Experiments were replicated two (c-g,k-o,q), three (p,r) or four (h-j,s) times from biologically independent samples with similar results. 1

S U P P L E M E N TA R Y I N F O R M AT I O N Supplementary Figure 2 (associated to Figure 2). Cpeb4 deletion causes mitochondrial dysfunction and defective lipid metabolism in hepatocytes.

2 S U P P L E M E N TA R Y I N F O R M AT I O N Supplementary Figure 2 (associated to Figure 2). Cpeb4 deletion causes mitochondrial dysfunction and defective lipid metabolism in hepatocytes. a, Cpeb4 mrna expression in livers from WT (n=8) and Cpeb4LKO (n=8) mice. Two-sided Student's t test, P= b, Immunoblot displaying CPEB4 and α-tubulin protein levels in WT and Cpeb4LKO mice. Unprocessed scans of original blots are shown in Supplementary Fig.7h. c, Weight evolution of WT (n=22) and Cpeb4LKO (n=30) mice fed standard diet. Two-way ANOVA, P= d, Glucose tolerance test after overnight fasting in WT (n=16) and Cpeb4LKO (n=16) mice. Two-way ANOVA, P= e, Plasma alanine aminotransferase levels of WT (n=12) and Cpeb4LKO (n=12) mice fed standard diet. Two-sided Student's t test, P= f-g, Liver weight (f) and hepatic triglyceride content (g) of WT and Cpeb4LKO mice fed HFD. Panel f: WT, n=44; Cpeb4LKO, n=44 mice. Panel g: WT-CHOW, n=12; WT-HFD, n=18; Cpeb4LKO-CHOW, n=20; Cpeb4LKO-HFD, n=18 mice. Twoway ANOVA, **P= (f), ***P=0.017 (g). h, Photograph of the liver, and H&E and Oil Red O staining of liver sections from the same animals. Representative images of 20 independent experiments are shown. Scale bar, 100 μm. i, Growth curve of WT (n=44) and Cpeb4LKO (n=48) mice on HFD. Two-way ANOVA, P= j, Fasn and Scd1 gene expression analysis by qrt-pcr of livers from WT (n=16) or Cpeb4LKO (n=16) mice. Two-way ANOVA, P= k, Analysis of palmitate uptake in primary hepatocytes; n=18 biologically independent dishes per group. Two-sided Student's t test, P= l, Immunoblot for the indicated mitochondrial markers and loading controls in WT and Cpeb4KO liver extracts, n=3 biologically independent samples. Unprocessed scans of original blots are shown in Supplementary Fig.7i. m, mtdna quantification normalized to nuclear DNA content by qrt-pcr of livers from WT (n=16) and Cpeb4KO (n=16) mice. Two-sided Student's t test, P= For a, c-g, i-k and m, data are mean±s.e.m. Experiments were replicated two (a,c-e,g,j), three (k) or four (f,i) times from biologically independent samples with similar results. 2

3 S U P P L E M E N TA R Y I N F O R M AT I O N Supplementary Figure 3 (associated to Figure 4). CPEB4 depletion leads to defective adaptation to chronic ER-stress. a, Apoptosis analysis of WT and Cpeb4KO MEFs measured by flow cytometry as the percentage of annexin V-positive cells after treatment with H202 (100 μm) or ionizing radiation (IR) (5 Gy) for 24 h; n=4 biologically independent MEF cell lines. Two-way ANOVA, P= b, Left: TUNEL staining of liver sections of WT and Cpeb4LKO mice injected with 1 mg/kg TM and killed 48h later. Scale bar, 100 μm. Arrows indicate apoptotic cells. Right: Quantification of the number of apoptotic cells in livers from WT (n=20) and Cpeb4LKO (n=20) mice. Twosided Student's t test, *P= Data are mean±s.e.m. Experiments in a,b were replicated two times from biologically independent samples with similar results. 3

4 Supplementary Figure 4 (associated to Figures 5 and 6). CPEB4 synthesis and translation of CPE-regulated mrnas are upregulated by UPR. a, Atf4 mrna analysis in WT MEFs treated with 1 μm thapsigargin (TG) for the indicated times. b, qrt-pcr expression analysis of the different luciferase constructs in HepG2 cells treated with 0.1 μm TG for 6h; n=18 biologically independent dishes. Two-way ANOVA, P= c, Left: Immunoblot for the indicated proteins in WT or Perk KO MEFs treated with 1,5 μm TG and harvested at the indicated times. Right: Immunoblot quantification. Unprocessed scans of original blots are shown in Supplementary Fig.7j. d, Total translation of Txnip assessed by ribosome profiling in MEFs treated with 1 μm TG for the indicated times (Reid D.W. et al., 2014). e, Txnip 3'UTR sequence in various mammalian species. Conserved CPE-elements are highlighted. f, Left: Txnip mrna poly(a) tail length quantification by epat assay in WT and Cpeb4 KO MEFs treated with TG for 2h. Right: Quantification of the area under the curve (AU); n=8 biologically independent MEF cell lines. Two-way ANOVA, *P= Data are mean±s.e.m. in a,c,f and mean±s.d. in b. Experiments were replicated two (c,f) or three (b) times from biologically independent samples with similar results. 4

5 Supplementary Figure 5 (associated to Figure 7). uorfs and CPEs determine mrna activation kinetics, which is influenced by the circadian clock. a, Gene expression analysis by qrt-pcr of Bmal1 and Per2 in WT and Cpeb4 KO mouse livers at the indicated ZT. Two-way ANOVA, P=0.95. b, Cpeb4 mrna levels in livers of WT fed mice at the indicated ZTs. 5

6 P eif2α PERK eif2α ER STRESS AAAA General translation inhibition Early activated mrnas (uorfs) uorf uorf Protein AAAA uorf uorf CPE CPEB4 mrna CPEB4 CPE AAAA Late activated mrnas (CPEs) Protein CPE CPE AAAAAAAAA Supplementary Figure 6 Working model: Sequential waves of translational activation during ER-stress mediated by PERK/uORFs and CPEB4/CPEs. The UPR triggers general translation inhibition. However, mrnas harbouring uorfs in their 5 UTRs are translationally activated at early time points after ER-stress, including Cpeb4 mrna. When CPEB4 is produced, it activates the translation of CPE-containing mrnas at late time points generating a second wave of protein production. 6

Supplementary Figure 7 Unprocessed scans of originals blots. a, Western blot corresponding to Fig.3a. b, Western blot corresponding to Fig.3e. c, Western blot corresponding to Fig.5a.

7 Supplementary Figure 7 Unprocessed scans of originals blots. a, Western blot corresponding to Fig.3a. b, Western blot corresponding to Fig.3e. c, Western blot corresponding to Fig.5a. d, Western blot corresponding to Fig.6b. e, Western blot corresponding to Fig.7d. f, Western blot corresponding to Fig.7e. g, Western blot corresponding to Supplementary Fig.1a. h, Western blot corresponding to Supplementary Fig.2b. i, Western blot corresponding to Supplementary Fig.2l. j, Western blot corresponding to Supplementary Fig.4c. 7

8 Supplementary Table Legends Supplementary Table 1 RNA-immunoprecipitation analysis showing mrnas specifically associated with CPEB4 in hepatocytes. Supplementary Table 2 mrnas showing reduced localization to endoplasmic reticulum in Cpeb4 KO livers. Supplementary Table 3 Putative uorfs contained withing rodent Cpeb4 5'UTR. Supplementary Table 4 Primer sequences used for PCR. 8

Supplementary Figure S I: Effects of D4F on body weight and serum lipids in apoe -/- mice.

Supplementary Figures: Supplementary Figure S I: Effects of D4F on body weight and serum lipids in apoe -/- mice. Male apoe -/- mice were fed a high-fat diet for 8 weeks, and given PBS (model group) or