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1 Van Nostrand Et Al SUPPLEMENTAL Van Nostrand et al SUPPLEMENTAL MATERIAL In this PDF: Supplemental Figures S1 -8 Supplemental Data Table 2 Supplemental Materials and Methods As a separate Excel File: Supplemental Data Table 1 – contains gene lists from all Figure panels 1 A B INK - Down-regulated mRNAs FC>1.5 FDR<0.05 WT Metformin WT WT Insulin WT WT Control WT Met + Ins WT WT INK WT WT Ins WT InsMet 1 INK induces, Metformin Independent (e.g. Lipid signaling, Autophagy) Met 658 2 INK and Met-repress (same) (Interferon-beta, Pyruvate 395 metabolism) 1051 InsInk Ink 62 89 3 INK induces, Met represses (opposite) (e.g. Macroautophagy) 480 93 76 876 4 INK-Independent, Insulin represses (Inflammation, Nutrient sensing) 104 16 INK and Met-induce (same) (IL-17 signaling, Lipid metabolism) 25 5 44 37 INK represses, Ins represses, Met Induces in Ins (opposite) (e.g. 365 6 Monocarboxylic acid metabolism, Amino acid biosynthesis, Steroid metabolism) 7 INK induces, Ins Induces, Met represses in Ins (opposite) (e.g. Protein localization) 8 INK and Met-repress (same) (Amino acid metabolism, Lipid Metabolism, HIF-1, ATF4) Upregulated mRNAs 9 INK-Independent, Insulin represses, Met Induces in Ins (e.g. Infection pathway) FC>1.5 FDR<0.05 10 INK and Met-repress (same) (e.g. RNA biology) Met InsMet 350 922 588 InsInk 11 INK-Independent, Met induces (Apoptosis, MAPK signaling) Ink 38 177 180 78 1192 12 INK-Independent, Met represses (e.g. Cell cycle) 41 142 22 5 64 65 475 C Ctrl vs Met Ins vs InsMet NES: -1.86 NES: -1.77 FDR: 0.0024 FDR: 0.0066 Ctrl Ins Met InsMet Supplemental Figure S1 A) Heatmap of genes regulated by metformin (Met) or INK-128 (0.1µM, INK) at 5 hours in the absence or with 15’ pretreatment of 1nM Insulin (Ins) with a fold change of at least 1.5. Clusters annotated for metformin (Met), INK- 128 (INK), and Insulin (Ins) responsiveness and primary biological process contained in signature according to metascape analysis. B) Venn Diagram of genes regulated in primary hepatocytes by 2mM metformin (Met) and 0.1mM INK-128 (INK) in the presence or absence of 1nM Insulin pre-treatment (Ins) as described in (A). Fold change>1.5, FDR<0.05 C) GSEA analysis of genes regulated by metformin in absence (left) or presence (right) of insulin pre-treatment as described in (A) compared with gene set for AKT-mTOR signaling showing enrichment for mTOR down upon treatment with metformin. +/+ AA/AA A B 2 hr 5 hr 2 hr 5 hr G C T Alanine G C G Alanine Met (mM) 0 0.5 1 2 0 0.5 1 2 0 0.5 1 2 0 0.5 1 2 Serine Serine P-722 RAPTOR T C C T C C P-792 RAPTOR RAPTOR AMPK Targets P-AMPK P-T389 S6K1 S6K1 Ser722Ala Ser792Ala P-S240/244 S6 Targets P-S235/236 S6 mTORC1 mTORC1 S6 4-EBP1 ACTIN C Primary Hepatocytes, Serum-Starved Up-regulated mRNAs Down-regulated mRNAs 220 260 76 117196 83 203 201 1000 1469 1387 998 (26%) (37%) (29%) (44%) AA WT AA AA WT WT AA WT Met Met/Ink Met InsMet Met&Ink InsMet&Ink InsMet InsMet/Ink D Genes Up-regulated with Metformin and Ink Genes Down-regulated with Metformin and Ink in WT but not in RaptorAA in WT but not in RaptorAA GO Biological Process GO Biological Process -log(p-value) -log(p-value) -log(p-value) -log(p-value) 0 1 2 3 0 1 2 3 4 0 1 2 3 0 1 2 3 4 Regulation of transcription Signal transduction Response to ER stress Cellular amino acid biosynthetic process Response to ER stress Cilium morphogenesis Pos reg of protein phosphorylation Negative reg of cell growth Neg Reg of protein kinase activity Glucose homeostasis ER Unfolded Protein Response Pos reg of ERK1/2 cascade Potassium ion transport Aging Immune system process Response to follicle-stimulating hormone Cell adhesion Cellular response to glucose stimulus Response to hypoxia Progesterone metabolic process Intracellular signal transduction Fat cell differentiation DNA binding transcription factor Apoptotic process Regulation of Rho protein signal transduction Transmembrane Transport Lipid metabolic process Regulation of TOR signaling KEGG Pathway Analysis -log(p-value) KEGG Pathway Analysis -log(p-value) 0 1 2 3 -log(p-value) 0.0 0.5 1.0 1.5 -log(p-value) Osteoclast differentiatin 0 1 2 3 0.0 0.5 1.0 1.5 African trypanosomiasis Staphylococcus aureus infection Protein processing in ER Bile secretion Leishmaniasis O-glycan Biosynthesis Biosynthesis of amino acid Axon guidance Estrogen signaling pathway Fructose and mannose metabolism E InsINK InsINK INK INK InsMet vs Ins vs Ins InsMet vs Ins vs Ins Met vs UT vs UT of Met vs UT vs UT WT AA T AT Am WT WT AA T AT Am WT WT AA T AT Am WT WT AA T AT Am WT Mlxipl Aldoart1 Lipid Naaa Metab Irs2 Aldoa Reg Gdf9 Crls1 Vcam1 metab Hk1 Sesn2 Pafah1b2 Glucose Fructose Fructose Response Neg Cell Growth Cell WT AA T AT Am WT WT AA T AT Am WT WT AA T AT Am WT WT AA T AT Am WT Alms1 Rasgrp1 Ero1l Adcy9 Diff Aloxe3 C5ar2 Dnajc3 Prkar2a Hmga2 Spry2 ER Dnaja4 ERK1/2 Fat Cell Fat Glucagon Stress Supplemental Figure S2 A) Chromatogram of sequence from Raptor Serine 722;Alanine 722 and Serine 792;Alanine 792 heterozygous alleles generated from CRISPR targeted mice. B) ) Immunoblot analysis of serum starved primary hepatocytes derived from wild-type (+/+) or RaptorAA mice treated with increasing doses of metformin (0, 0.5, 1, or 2mM) for 2 or 5 hours and blotted for AMPK and mTORC1 substrates C) Venn diagram depicting overlap between genes identified as being both metformin and INK-dependent and genes regulated in RaptorAA hepatocytes upon metformin in the absence or presence of insulin as described in Figure 1C. D) GO Term and KEGG pathway analysis of RaptorAA-dependent genes up-regulated (left) or down-regulated (right) by metformin and INK as described in Figure 1C by a fold change of at least 1.5 in wild-type primary hepatocytes and a fold change of less than 1.5 in RaptorAA primary hepatocytes in presence or absence of insulin. Length of Bar correlates to –log(p-value). Identified using DAVID software E) Example heatmap of fold change for genes identified as being AA-dependent (FC>1.5 with metformin and Ink in WT; FC<1.5 upon metformin in AA). A B Generation of Hepatocytes: Isolate Treat Cells Nomenclature Genotype Tam Treat Hepatocytes & Collect WT +/+ or Tsc2fl/fl or Ampka1fl/fl;Ampka2fl/fl S722A;S792A/S722A;S792A Day 1 3 5 AA Raptor Next Day In vivo Tsc2fl/fl;Alb-CreER/+ or 7-10 days later Tsc2 Tsc2fl/fl;Alb-Cre/+ (Tsc2-null) Hepatocyte Treatments: RaptorS722A;S792A/S722A;S792A;Tsc2fl/fl; Treat w/ Treat w/ Treat w/ Translation Transcriptome AA;Tsc2 or AT Alb-CreER/+ or Alb-Cre/+ Insulin Metformin/INK Puromycin Analysis Analysis 5h Ampk Ampka1fl/fl;Ampka2fl/fl;Alb-CreER/+ -15’ 0 1h 2h 50’ C D 1.0 1.5 1.5 1.5 **** WT **** 1.5 * * AA 0.8 *** **** **** T WT 1.0 1.0 * *** 1.0 0.6 AT AA S6 - S6 0.4 S6K1 P-S6 - - P 0.5 S6K1 T 0.5 - 0.5 P 1.0 0.2 P P AT (Treated/Untreated) Treated/ UntreatedTreated/ 0.0 UntreatedTreated/ 0.0 0.0 0.0 Treated/ UntreatedTreated/ Treated/ UntreatedTreated/ WT AA T AT WT AA T AT WT AA T AT WT AA T AT WT AA T AT WT AA T AT WT AA T AT WT AA T AT 0.5 N.S. +Ins N.S. +Ins N.S. +Ins N.S. +Ins 0.0 WT AA T AT WT AA T AT N.S. +Ins Supplemental Figure S3 A) Nomenclature for genotypes of mice used throughout the paper. Floxed alleles treated with tamoxifen (Tam) generates null alleles. B) Schematic of time course used to treat mice with tamoxifen prior to generation of hepatocytes (top) and time course used for treating hepatocytes for various assays used throughout the paper (bottom). C) Quantification of P-S6 and P-S6K relative to total protein in immunoblots from Figure 3C D) Quantification of P-S6K and P-S6 relative to total protein in immunoblots from Figure 3D Treat w/ Treat w/ Treat w/ Translation 2 Ink-Dependent Genes A Insulin Metformin/INK Puromycin Analysis F 1 -15’ 0 1h 2h 50’ +1nM Insulin P=0.2 B 1.0 * 0 WT AA T AA;T Ampk WT 0.8 Metformin - + - + - + - + - + Ink 0.6 -1 Anti-Puro 0.4 (Treated/Untreated) PuroIncorporation (Treated/Untreated) 0.2 ACTIN 0.0 -2 +/+ AA T AA;T Ampk +/+ Primary Hepatocytes, Insulin-Pretreatment + 2mM Metformin + Ink Efficiency Translation Change Fold + Insulin -3 **** 0.20 0.15 0.20 WT UT 80s +/+ +/+ AA T AA;T Ampk WT Met AA UT C WT Ink Tsc UT 80s 80S AA Met Tsc Met 0.15 80s 0.15 0.10 + Ink + 2mM Metformin Polysome 40s 60s 0.10 0.10 40s 60s Polysome Abs (254nm) Abs Abs (254nm) Abs Genes with Ink-Dependent Translation (190 genes) Abs (254nm) Abs Polysome 0.05 G Polysome 60s60S 4 GO Term Analysis 40S0 -log(p-value) 0.05 0.05 s 0 5 10 15 20 25 10 20 30 40 50 0.00 10 20 30 40 50 10 20 30 40 50 Translation Distance (mm) Distance (mm) Distance (mm) Transport Oxidation-reduction process 0.06 0.15 Ribosomal small subunit assembly 80s AATsc UT 80s rRNA processing AATsc Met AMPK UT AMPK Met 60s Cytoplasmic translation 0.04 40s 0.10 Polysome 40s 60s Polysome KEGG Pathway Analysis -log(p-value) Abs (254nm) Abs (254nm) Abs 0.02 0.05 0 10 20 30 40 Ribosome Oxidative phosphorylation 0.00 0.00 10 20 30 40 50 10 20 30 40 50 Distance (mm) Distance D 3 E Metformin-Dependent Genes H All Genes 2 TOP-Motif Genes 2 GO Biological Processes -log(p-value) 1 0 5 10 15 1 Translation Maturation of SSU-rRNA from 0 0 tricistronic rRNA transcript Oxidation-reduction process rRNA processing -1 -1 (Treated/Untreated) Ribosomal small subunit biogenesis (Treated/Untreated) * -2 -2 ** Fold Change Translation Efficiency Efficiency Translation Change Fold KEGG Pathways ** -log(p-value) Efficiency Translation Change Fold ** -3 * 0 5 10 15 20 +/+ +/+ AA T AA;T Ampk -3 *** Ribosome +/+ +/+ AA T AA;T Ampk + Ink + 2mM Metformin Oxidative phosphorylation + Ink + 2mM Metformin Parkinson's disease Alzheimer's disease Huntington's disease Non-alcoholic fatty liver disease Supplemental Figure S4 A) Schematic of time course used to assay hepatocytes for translation.
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