SIRT6 histone deacetylase functions as a potential oncogene in human melanoma -

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1 SIRT6 histone deacetylase functions as a potential oncogene in human melanoma - Garcia-Peterson et al Supplementary Figure S:Tissue microarray (TMA) staining and organization. A) Diagram of the TMA indicating location and tissue type of each core. B) Whole image of the SIRT6- stained slide with hematoxylin counterstaining. ME004c indicates the catalog number from US Biomax, Inc that was used for the study.

2 Supplementary Figure S2: Multiple SIRT6-targeting shrna constructs were analyzed for SIRT6 knockdown. (A) A375 melanoma cells were transfected using Lipofectamine 2000 using manufacturer s protocol (using 4 μg of DNA and 0 μl of Lipofectamine reagent per well in six well plate) with nonsense (NS) and five SIRT6-targeting shrna for 48 h, and cells were analyzed 48 h later. Cell lysates were subjected to SDS-PAGE and western blotting for SIRT6 protein as in Materials and Methods. Equal loading was confirmed by reprobing the blot for β- tubulin. Further, the two best constructs were chosen to determine which had the best knockdown via lentiviral transduction. (B) A375 cells were transduced with nonsense (NS) and the two best SIRT6 knockdown constructs from A, -73 and -76, for 48 h and collected 48 h later. Cell lysates were subjected to western blot and RT-qPCR to confirm SIRT6 knockdown. β- tubulin and GAPDH were used as controls, respectively.

3 Supplementary Table S: Quantification of SIRT6 staining and description of samples. Type Sex Age Organ Pathology Stage TNM Position Nuclear SIRT6 Mean Cytoplasmic SIRT6 Mean Whole Cell SIRT6 Mean Malignant M Adrenal 42 gland Pheochromocytoma (tissue marker) Benign F 2 Skin Intradermal nevus of right leg J Benign M 38 Skin Compound nevus of right face I Benign M 25 Skin Compound nevus of left leg I Benign F 29 Skin Compound nevus of left shoulder I Benign F 30 Skin Intradermal nevus of left rump I Benign M 20 Skin Compound nevus of left shoulder J Benign M 39 Skin Intradermal nevus of head J Benign F 34 Skin Sebaceous nevus of head (skin tissue) J Benign F 23 Skin Compound nevus of right lumbar part I Benign F 2 Skin Junctional nevus of neck I Benign M 50 Skin Compound nevus of upper arm J Benign M 8 Skin Sebaceous nevus of left frontal region (skin tissue) J Benign M 6 mo Skin Intradermal nevus of face I Benign M Skin Intradermal nevus of left thigh J Benign M 42 Skin Intradermal nevus of face J Benign M 73 Skin Compound nevus of left heel J Benign F 0 Skin Junctional nevus of right foot I Benign M 55 Skin Compound nevus of back J Malignant M 7 Skin Malignant melanoma of right rump II T4N0M0 A Malignant F 38 Vulva Malignant melanoma I E Malignant M 60 Skin Malignant melanoma of right rump III T4NM0 A Malignant F 4 Skin Malignant melanoma of scalp II T4N0M0 C Malignant F 32 Skin Malignant melanoma of right lumbar part (fibrofatty tissue) II T4N0M0 A Malignant M 79 Skin Malignant melanoma of left face I TN0M0 B Malignant F 38 Rectum Malignant melanoma I E Malignant M 55 Skin Malignant melanoma of left sole II T4N0M0 A Malignant M 64 Rectum Malignant melanoma II E Malignant F 46 Skin Malignant melanoma of right thumb II T4N0M0 D Malignant M 55 Stomach Malignant melanoma I F Malignant M 6 Skin Malignant melanoma of left armpit IV T4bN0M A Malignant M 3 Skin Malignant melanoma of scalp II T4N0M0 D Malignant M 65 Skin Malignant melanoma of scalp II T4N0M0 D Malignant F 44 Vulva Malignant melanoma I E Malignant M 4 Skin Malignant melanoma of left leg II T3N0M0 B Malignant M 49 Skin Malignant melanoma of left arm II T4N0M0 C Malignant F 72 Rectum Malignant melanoma I F Malignant M 55 Stomach Malignant melanoma I F Malignant F 52 Rectum Malignant melanoma I E Malignant F 47 Skin Malignant melanoma of left upper arm II T4N0M0 D Malignant M 5 Skin Malignant melanoma of back II T4N0M0 A Malignant F 84 Rectum Malignant melanoma of crissum I E Malignant F 67 Rectum Malignant melanoma I E Malignant F 42 Skin Malignant melanoma of right thigh II T4N0M0 B Malignant M 75 Rectum Malignant melanoma II F Malignant F 72 Vulva Malignant melanoma I D Malignant melanoma of left shoulder Malignant M 50 Skin (fibrous tissue and blood vessel) II T4N0M0 B Malignant F 83 Skin Malignant melanoma of right little finger I T2N0M0 C Malignant M 6 Skin Malignant melanoma of right groin III T4N2M0 B Malignant M 25 Skin Malignant melanoma of left sole I T2N0M0 A Malignant F 66 Rectum Malignant melanoma I F Malignant M 42 Skin Malignant melanoma of left heel IV T3N2M C Malignant M 80 Skin Malignant melanoma of right sole II T4N0M0 A Malignant M 37 Skin Malignant melanoma of right upper arm II T4N0M0 B Malignant M 72 Skin Malignant melanoma of left sole IB T2aN0M0 A Malignant M 52 Skin Malignant melanoma of left abdominal wall II T4N0M0 B Malignant M 50 Esophagus Malignant melanoma II F Malignant M 73 Intestine Malignant melanoma I G

4 Continued from previous page Malignant M 4 Skin Malignant melanoma of left forearm II T4N0M0 C Malignant M 7 Skin Malignant melanoma of right groin II T4N0M0 C Malignant F 54 Rectum Malignant melanoma of anal tube II F Malignant M 66 Rectum Malignant melanoma I F Malignant M 45 Skin Malignant melanoma of crissum II T4N0M0 D Malignant M 66 Skin Malignant melanoma of right thigh II T4N0M0 C Malignant F 59 Skin Malignant melanoma with necrosis of anus II T4N0M0 B Malignant M 5 Skin Malignant melanoma of left armpit II T4N0M0 C Malignant M 5 Skin Malignant melanoma of left upper arm III T4NM0 C Malignant F 46 Skin Malignant melanoma of thigh II T4N0M0 A Malignant F 62 Vulva Malignant melanoma I E Malignant F 45 Skin Malignant melanoma of left thigh I T2N0M0 D Malignant F 57 Vulva Malignant melanoma I D Malignant F 72 Skin Malignant melanoma of right cheek II T4N0M0 D Malignant M 40 Skin Malignant melanoma of right chest wall II T4N0M0 B Malignant F 44 Rectum Malignant melanoma I E Malignant M 7 Intestine Malignant melanoma I F Malignant F 45 Vulva Malignant melanoma I E Malignant M 64 Esophagus Malignant melanoma I F Malignant F 59 Skin Malignant melanoma of right rump I TN0M0 B Malignant melanoma of left parotid Malignant F 70 Oral cavity gland I G Malignant M 49 Skin Malignant melanoma of left foot III T4NM0 D Malignant M 56 Skin Malignant melanoma of abdominal wall II T4N0M0 C node groin H node Malignant malignant melanoma from right armpit G node left groin G node right groin (fibrous tissue and blood vessel) I node thigh H node Malignant malignant melanoma from right armpit H node Malignan tmalignant melanoma from left sole H node left groin H node right groin H node right groin H node right heel G node right groin G node neck I node left ear H node left thump G node right groin G node left groin G node right leg H node left Lower gum G node left groin H "-" indicates fields where data was not available or not provided. Samples without a staining intensity were eliminated because of either lack of sufficient melanocytes or poor section quality for tissue segmentation.

5 Supplementary Table S2: Details of shrna vectors used Vector ID Clone ID Sequence Control Vector (shns) N/A CCGGCAACAAGATGAAGAGCACCAACTCGAGTTGGTGCT CTTCATCTTGTTGTTTTT TRCN (shsirt6-73) NM_ sc CCGGTGGAAGAATGTGCCAAGTGTACTCGAGTACACTTG GCACATTCTTCCATTTTTG TRCN (shsirt6-74) NM_ sc CCGGGCAGTCTTCCAGTGTGGTGTTCTCGAGAACACCAC ACTGGAAGACTGCTTTTTG TRCN (shsirt6-75) NM_ sc CCGGGCTGGGTACATCGCTGCAGATCTCGAGATCTGCAG CGATGTACCCAGCTTTTTG TRCN (shsirt6-76) NM_ sc CCGGCACCCGGATCAACGGCTCTATCTCGAGATAGAGCC GTTGATCCGGGTGTTTTTG TRCN (shsirt6-77) NM_ sc CCGGCACGGGAACATGTTTGTGGAACTCGAGTTCCACAA ACATGTTCCCGTGTTTTTG

6 Supplementary Table S3: Primer sequences used for RT-qPCR validation. Gene Amplicon Primer Length Tm Primer Bank Primer Sequence (5' - > 3') size (bp) orientation (bp) ( C) ID SIRT6 94 F CCCACGGAGTCTGGACCAT c R CTCTGCCAGTTTGTCCCTG 9 60 AKT 96 F AGCGACGTGGCTATTGTGAAG c R GCCATCATTCTTGAGGAGGAAGT ATG0 80 F ATAAGATGCGACTGCTACAGGG c3 R CAATGCTCAGCCATGATGTGAT 22 6 ATG2 53 F TAGAGCGAACACGAACCATCC c2 R CACTGCCAAAACACTCATAGAGA ATG3 25 F GATGGCGGATGGGTAGATACA c3 R TCTTCACATAGTGCTGAGCAATC ATG7 4 F ATGATCCCTGTAACTTAGCCCA c3 R CACGGAAGCAAACAACTTCAAC 22 6 BAK 92 F CATCAACCGACGCTATGACTC c3 R GTCAGGCCATGCTGGTAGAC BCL2L 79 F GACTGAATCGGAGATGGAGACC c2 R GCAGTTCAAACTCGTCGCCT CLN3 24 F CGCCCACGACATCCTTAGC c R AGCAGCCGTAGAGACAGAGTT 2 63 CTSB 59 F AGAGTTATGTTTACCGAGGACCT c3 R GATGCAGATCCGGTCAGAGA 20 6 CTSS 3 F TGACAACGGCTTTCCAGTACA c2 R GGCAGCACGATATTTTGAGTCAT 23 6 DRAM2 36 F CTGTGCTTACCTTTGGTATGGG c R GCACTTACTCCACACCAGATAAC 23 6 GAA 29 F TGCCCTCGCAGTATATCACAG c3 R GAGACCCGTAGAGGTTCGC 9 62 HSP90AA 87 F GCTTGACCAATGACTGGGAAG c3 R AGCTCCTCACAGTTATCCATGA IRGM 89 F GCCATGAATGTTGAGAAAGCCT c R GTCCTGGACACTATCTTCAGAGT 23 6 NPC 89 F GTCCAGCGCAGGTGTTTTC c R GCCGAACATCACAACAGAGAC 2 6 SQSTM 6 F AAGCCGGGTGGGAATGTTG c3 R CCTGAACAGTTATCCGACTCCAT 23 6 TNFα 9 F GAGGCCAAGCCCTGGTATG c2 R CGGGCCGATTGATCTCAGC 9 63 GAPDH 0 F CTGGGCTACACTGAGCACC c3 R AAGTGGTCGTTGAGGGCAATG 2 63

7 Supplementary Table S4: Human Autophagy PCR Array genes modulated upon SIRT6 knockdown in A375 melanoma cells. Gene Gene Description Autophagy Machinery Components Fold Change AKT V-akt murine thymoma viral oncogene homolog Co-Regulators of Autophagy and Apoptosis -2 AMBRA Autophagy/beclin- regulator Involved in Autophagic Vacuole Formation APP Amyloid beta (A4) precursor protein Co-Regulators of Autophagy and Apoptosis ATG0 ATG0 autophagy related 0 homolog Responsible for Protein Transport 2.03 ATG2 ATG2 autophagy related 2 homolog Involved in Autophagic Vacuole Formation, Co-Regulators of -2 Autophagy and Apoptosis ATG6L ATG6 autophagy related 6-like Involved in Autophagic Vacuole Formation, Responsible for.0 Protein Transport ATG6L2 ATG6 autophagy related 6-like 2 Responsible for Protein Transport -.59 ATG3 ATG3 autophagy related 3 homolog Involved in Protein Ubiquitination, Responsible for Protein -2 Transport ATG4A ATG4 autophagy related 4 homolog A Involved in Autophagic Vacuole Formation, Responsible for Protein Targeting to Membrane / Vacuole, Responsible for Protein Transport, Gene with Protease Activity ATG4B ATG4 autophagy related 4 homolog B Involved in Autophagic Vacuole Formation, Responsible for Protein Targeting to Membrane / Vacuole, Responsible for Protein Transport, Gene with Protease Activity ATG4C ATG4 autophagy related 4 homolog C Involved in Autophagic Vacuole Formation, Responsible for - Protein Targeting to Membrane / Vacuole, Responsible for Protein Transport, Gene with Protease Activity ATG4D ATG4 autophagy related 4 homolog D Involved in Autophagic Vacuole Formation, Responsible for - Protein Targeting to Membrane / Vacuole, Responsible for Protein Transport, Gene with Protease Activity ATG5 ATG5 autophagy related 5 homolog Involved in Autophagic Vacuole Formation, Co-Regulators of Autophagy and Apoptosis ATG7 ATG7 autophagy related 7 homolog Responsible for Protein Transport, Involved in Protein -2 Ubiquitination ATG9A ATG9 autophagy related 9 homolog A Involved in Autophagic Vacuole Formation, Responsible for Protein Transport ATG9B ATG9 autophagy related 9 homolog B Involved in Autophagic Vacuole Formation -.26 BAD BCL2-associated agonist of cell death Co-Regulators of Autophagy and Apoptosis BAK BCL2-antagonist/killer Co-Regulators of Autophagy and Apoptosis -2.0 BAX BCL2-associated X protein Co-Regulators of Autophagy, Apoptosis and the Cell Cycle BCL2 B-cell CLL/lymphoma 2 Co-Regulators of Autophagy and Apoptosis -.58 BCL2L BCL2-like Co-Regulators of Autophagy and Apoptosis -2 BECN Beclin, autophagy related Co-Regulators of Autophagy and Apoptosis, Involved in - Autophagic Vacuole Formation BID BH3 interacting domain death agonist Co-Regulators of Autophagy and Apoptosis -.58 BNIP3 BCL2/adenovirus EB 9kDa interacting protein 3 Co-Regulators of Autophagy and Apoptosis -.58 CASP3 Caspase 3, apoptosis-related cysteine peptidase Co-Regulators of Autophagy and Apoptosis CASP8 Caspase 8, apoptosis-related cysteine peptidase Co-Regulators of Autophagy and Apoptosis - CDKNB Cyclin-dependent kinase inhibitor B (p27, Kip) Co-Regulators of Autophagy, Apoptosis and the Cell Cycle -.6 CDKN2A Cyclin-dependent kinase inhibitor 2A (melanoma, Co-Regulators of Autophagy, Apoptosis and the Cell Cycle - p6, inhibits CDK4) CLN3 Ceroid-lipofuscinosis, neuronal 3 Co-Regulators of Autophagy and Apoptosis -2 CTSB Cathepsin B Co-Regulators of Autophagy and Apoptosis -2.0 CTSD Cathepsin D Autophagy in Response to Other Intracellular Signals -.0 CTSS Cathepsin S Autophagy in Response to Other Intracellular Signals -4 CXCR4 Chemokine (C-X-C motif) receptor 4 Co-Regulators of Autophagy and Apoptosis DAPK Death-associated protein kinase Co-Regulators of Autophagy and Apoptosis DRAM DNA-damage regulated autophagy modulator Co-Regulators of Autophagy and Apoptosis, Gene Linking Autophagosome to Lysosome DRAM2 DNA-damage regulated autophagy modulator 2 Autophagy in Response to Other Intracellular Signals -2 EIF2AK3 Eukaryotic translation initiation factor 2-alpha Co-Regulators of Autophagy and Apoptosis, Autophagy.0 kinase 3 Induction by Intracellular Pathogens EIF4G Eukaryotic translation initiation factor 4 gamma, Autophagy in Response to Other Intracellular Signals ESR Estrogen receptor Autophagy in Response to Other Intracellular Signals FADD Fas (TNFRSF6)-associated via death domain Co-Regulators of Autophagy and Apoptosis -.59 FAS Fas (TNF receptor superfamily, member 6) Co-Regulators of Autophagy and Apoptosis - GAA Glucosidase, alpha; acid Autophagy in Response to Other Intracellular Signals 2.0 GABARAP GABA(A) receptor-associated protein Involved in Autophagic Vacuole Formation, Responsible for Protein Transport, Gene Linking Autophagosome to Lysosome

8 Continued from previous page GABARAPL GABA(A) receptor-associated protein like Involved in Autophagic Vacuole Formation, Responsible for Protein Transport GABARAPL2 GABA(A) receptor-associated protein-like 2 Involved in Autophagic Vacuole Formation, Responsible for Protein Transport -.57 HDAC Histone deacetylase Co-Regulators of Autophagy and Apoptosis HDAC6 Histone deacetylase 6 Involved in Protein Ubiquitination HGS Hepatocyte growth factor-regulated tyrosine Autophagy in Response to Other Intracellular Signals kinase substrate HSP90AA Heat shock protein 90kDa alpha (cytosolic), Chaperone-Mediated Autophagy class A member HSPA8 Heat shock 70kDa protein 8 Chaperone-Mediated Autophagy - HTT Huntingtin Co-Regulators of Autophagy and Apoptosis IFNG Interferon, gamma Co-Regulators of Autophagy, Apoptosis and the Cell Cycle, Autophagy Induction by Intracellular Pathogens IGF Insulin-like growth factor (somatomedin C) Co-Regulators of Autophagy and Apoptosis INS Insulin Co-Regulators of Autophagy and Apoptosis IRGM Immunity-related GTPase family, M Involved in Autophagic Vacuole Formation LAMP Lysosomal-associated membrane protein Gene Linking Autophagosome to Lysosome, Autophagy.0 Induction by Intracellular Pathogens MAPLC3A Microtubule-associated protein light chain 3 Involved in Autophagic Vacuole Formation alpha MAPLC3B Microtubule-associated protein light chain 3 Involved in Autophagic Vacuole Formation.02 beta MAPK4 Mitogen-activated protein kinase 4 Autophagy in Response to Other Intracellular Signals - MAPK8 Mitogen-activated protein kinase 8 Co-Regulators of Autophagy and Apoptosis - MTOR Mechanistic target of rapamycin Co-Regulators of Autophagy and Apoptosis (serine/threonine kinase) NFKB Nuclear factor of kappa light polypeptide gene Co-Regulators of Autophagy and Apoptosis -.26 enhancer in B-cells NPC Niemann-Pick disease, type C Gene Linking Autophagosome to Lysosome -2 PIK3C3 Phosphoinositide-3-kinase, class 3 Autophagy in Response to Other Intracellular Signals PIK3CG Phosphoinositide-3-kinase, catalytic, gamma Co-Regulators of Autophagy and Apoptosis -.04 polypeptide PIK3R4 Phosphoinositide-3-kinase, regulatory subunit 4 Autophagy in Response to Other Intracellular Signals PRKAA Protein kinase, AMP-activated, alpha catalytic Co-Regulators of Autophagy and Apoptosis subunit PTEN Phosphatase and tensin homolog Co-Regulators of Autophagy, Apoptosis and the Cell Cycle RAB24 RAB24, member RAS oncogene family Responsible for Protein Transport RB Retinoblastoma Co-Regulators of Autophagy and the Cell Cycle RGS9 Regulator of G-protein signaling 9 Involved in Autophagic Vacuole Formation RPS6KB Ribosomal protein S6 kinase, 70kDa, Autophagy in Response to Other Intracellular Signals - polypeptide SNCA Synuclein, alpha (non A4 component of amyloid Co-Regulators of Autophagy and Apoptosis.28 precursor) SQSTM Sequestosome Co-Regulators of Autophagy and Apoptosis -2 TGFB Transforming growth factor, beta Co-Regulators of Autophagy, Apoptosis and the Cell Cycle TGM2 Transglutaminase 2 (C polypeptide, proteinglutamine-gamma-glutamyltransferase) Co-Regulators of Autophagy and Apoptosis TMEM74 Transmembrane protein 74 Autophagy in Response to Other Intracellular Signals -.59 TNF Tumor necrosis factor Co-Regulators of Autophagy and Apoptosis TNFSF0 Tumor necrosis factor (ligand) superfamily, Co-Regulators of Autophagy and Apoptosis -.03 member 0 TP53 Tumor protein p53 Co-Regulators of Autophagy, Apoptosis and the Cell Cycle ULK Unc-5-like kinase (C. elegans) Involved in Autophagic Vacuole Formation ULK2 Unc-5-like kinase 2 (C. elegans) Autophagy in Response to Other Intracellular Signals UVRAG UV radiation resistance associated gene Autophagy in Response to Other Intracellular Signals WIPI WD repeat domain, phosphoinositide interacting Involved in Autophagic Vacuole Formation