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Supplementary Information A Generation of βKcnj11-/- mice 5’ untranslated 3’ untranslated Wild allele C.R Kcnj11fl/fl RIP-Cre+ C.R FRT FRT 3’ arm 5’ arm BGH IoxP+Neo allele C.R PGK Neo promoter polyA RIPCre+;Kcnj11fl/+ Kcnj11fl/fl fl/fl fl/fl 5’ arm 3’ arm Kcnj11 RIP-Cre+;Kcnj11 Floxed (fl) allele C.R 5’ arm 3’ arm Null (-) allele RIP-Cre+;Kcnj11fl/fl Kcnj11fl/fl C.R: Coding region (βKcnj11-/-) (Control) B Gene expression C Islet morphology Kcnj11fl/fl Kcnj11fl/fl βKcnj11-/- βKcnj11-/- Insulin/Glucagon/DAPI 2.5 1.6 n.s. fl n.s. fl/ 1.4 n.s. 2.0 1.2 ** 1.0 Kcnj11 gene expression 1.5 0.8 -/- 0.6 1.0 0.4 Kcnj11 0.5 0.2 Kcnj11 β (fold change of control) (fold change of control) Relative Relative gene expression 0.0 0.0 Scale bars: 100 μm Kcnj11 Abcc8 Ins1 Ins2 Heart Skeletal Brain Relative Relative muscle D Insulin content E ipGTT fl/fl ** Kcnj11fl/fl 600 Kcnj11 0.8 * -/- -/- n.s ** βKcnj11 ** βKcnj11 500 . 500 ** 0.6 n.s 400 ) 400 ** dL n.s 300 300 ** 0.4 * 200 (mg/ 200 Blood Blood glucose 0.2 ng/5 islets) Insulin content ( 100 100 Plasma insulin (ng/mL) Plasma 0 0 0 Kcnj11fl/fl βKcnj11-/- 0 30 60 90 120 0 15 30 0 15 30 Time (min) Time (min) F oGTT G Perfusion of pancreas H Glucagon Kcnj11fl/fl Veh fl/fl ) Tolb/Veh Kcnj11 Tolb 2.8 mM glucose fl/fl βKcnj11-/- Veh Kcnj11 500 100 μM tolbutamide 8 βKcnj11-/- βKcnj11-/- Tolb 10 n.s. 400 6 8 n.s. n.s. 300 Kcnj11fl/fl 6 βKcnj11-/- /5 islets) /5 4 200 pg 4 ( * 2 Blood Blood glucose (mg/dL 100 2 secretionGlucagon n.s. ** * * * 0 0 0 -30 0 30 60 90 120 Insulin secretion (ng/mL) 0 5 10 15 20 25 Glucose (mM) 1.67 16.7 8.4 8.4 Time (min) Time (min) Arginine (20 mM) + - Carbachol (100 µM) - + 1 Oduori et al. Supplementary Figure 1. (A) Generation of Kcnj11-/- mice. Left, Schematic diagram of Kcnj11 gene targeting. The targeting vector containedβ phosphoglycerol kinase (PGK) promoter/a neomycin (Neo) resistant selection cassette flanked by flippase recognition target (FRT) sites and loxP sites on either side of the exon of the Kcnj11 gene. The neomycin resistant (Neor) cassette (flanked by FRT sites) was added to the targeting vector for positive selection, and was deleted in mice using FLP-FRT recombination system. Targeted allele, floxed allele after flippase recombination and final allele with the targeted region deleted are shown. Right, Production of bKcnj11-/- mice by crossing Kcnj11fl/fl mice with RIP-Cre+; Kcnj11fl/fl mice. (B) Expressions of Kcnj11, Abcc8, Ins1, and Ins2 genes in the islets and Kcnj11 in the brain, heart, and skeletal muscles assessed by qPCR (n = 3 for each). Expression of each gene was normalized by that of Gapdh and presented as fold change of control. (C) Immunohistochemistry of the islets. Green, insulin; Red, glucagon. (D) Insulin content of the islets (n = 16 for both groups). (E) Intraperitoneal(ip) glucose tolerance test. Left, blood glucose. Right, plasma insulin (n = 7 for both groups). (F) oGTT following tolbutamide (Tolb) administration. Tolb (50 mg/kg) was orally administered to 6 h-fasted mice 20 min prior to glucose challenge (1.5 g/kg) (n = 6 per group). Veh, vehicle (0.5% carboxymethyl cellulose). (G) Effects of Tolb on insulin secretion from perfused pancreases in the presence of 2.8 mM glucose. Basal insulin secretion in Kcnj11-/- mice was significantly elevated compared to control (n = 4 for both groups). β (H) Glucagon secretion from the islets at high glucose (16.7 mM), low glucose (1.67 mM), and arginine or carbachol at 8.4 mM glucose (n = 5-6 for each condition). Statistical analyses were performed by 2-tailed Student’s unpaired t-test for (B), (D), and (H) or by 2-way ANOVA followed by Dunnett’s post hoc test for (E), (F), and (G). Data represent mean ±SEM. *p < 0.05, **p < 0.01 2 A oGTT B oGTT C oGTT n.s. ** n.s. ** ** ** ** ** ** ) ) ) ** 3 * 3 3 ** AUC (X 10 AUC (X 10 AUC (X 10 Veh Sita Veh Sita Sita Sita + Sita Sita + Veh Lira Veh Lira Kcnj11fl/fl βKcnj11-/- Ex9 Ex9 Kcnj11fl/fl βKcnj11-/- Kcnj11fl/fl βKcnj11-/- D ipGTT Kcnj11fl/fl Veh n.s. 600 Kcnj11fl/fl GLP-1 -/- * ** ) βKcnj11 Veh 500 -/- * βKcnj11 GLP-1 ** ) 400 3 300 200 AUC (X 10 100 Blood Blood glucose (mg/dL 0 -30 0 30 60 90 120 Veh GLP-1 Veh GLP-1 Time (min) Kcnj11fl/fl βKcnj11-/- E ipGTT Kcnj11fl/fl Veh 600 Kcnj11fl/fl GIP ** ) βKcnj11-/- Veh * 500 βKcnj11-/- GIP ) ** 400 3 300 200 AUC (X 10 100 Blood Blood glucose (mg/dL 0 -30 0 30 60 90 120 Veh GIP Veh GIP Time (min) Kcnj11fl/fl βKcnj11-/- 2.8 mM G 16.7 mM G 2.8 mM G F Perfusion of pancreas 10 nM GIP 80 2.8 mM G 8.8 mM G 2.8 mM G 12 70 60 10 Kcnj11fl/fl βKcnj11-/- 50 Kcnj11fl/fl 8 βKcnj11-/- 40 6 30 4 20 2 Insulin secretion (ng/mL) 10 Insulin secretion (ng/mL) 0 0 0 15 20 25 30 35 0 5 10 15 20 25 30 35 Time (min) Time (min) 3 Oduori et al. Supplementary Figure 2. (A) AUC during oGTT following sitagliptin (Sita) administration in Figure 2A (n = 5-8 per group). (B) AUC during oGTT following sitagliptin (Sita) and exendin-9 (Ex9) administration in Figure 2D (n = 6-10 per group). (C) AUC during oGTT following liraglutide (Lira) administration in Figure 2E (n = 7-12 per group). (D) ipGTT following exogenous administration of GLP-1 (left) and AUC (right) (n = 6-8 per group). (E) ipGTT following exogenous administration of GIP (left) and AUC (right) (n = 6-14 per group). (F) Effect of glucose alone on insulin secretion from perfused pancreases (n = 4 for both groups). (G) Effect of high GIP concentration (10 nM) on insulin secretion from perfused pancreases (n = 4 for both groups). GLP-1 or GIP (100 µg/kg, each) was intraperitoneally administered to mice 20 min before glucose challenge in (D) and (E). Veh, vehicle (water); AUC, area under the curve. Mice were fasted overnight before perfusion experiments commenced in (F) and (G). Statistical analyses were performed by 2-way ANOVA followed by Tukey’s post hoc test. Data represent mean ±SEM. *p < 0.05, **p < 0.01 4 A Incretin receptors B Adenylyl cyclases C Phosphodiestrases -/- fl/fl -/- -/- fl/fl βKcnj11 Kcnj11 βKcnj11 Kcnj11fl/fl βKcnj11 Kcnj11 Gene expression Gene Gene expression Gene Gene expression Gene D Kcnj11 gene sequence Wild-type sequence TATCCCTGAGGAATATGTGCTGACCCGGCTGGCAGAGGACCCTGCAGAGCCCAGGTACCGTACTCGAGAGAGGAGGGCCCGCTTCGTG Kcnj11-/-βCL1 allele 1: -13 TATCCCTGAGAAATA ------------- CTGGCAGAGGACCCTGCAGAGCCCAGGTACCGTACTCGAGAGAGGAGGGCCCGCTTCGTG Kcnj11-/-βCL1 allele 2: +37 TATCCCTGAGGAATATGTGCTGACCCGGCTGGCAGAGGACCCTGCAGAGCCCAGGTACCGTACTCGAGAGAGGAGGGCCCGCTTCGTG GTGCTGACCCGGCTGGCAGAGGACCCTGCAGAGCCAG Kcnj11-/-βCL2 allele 1: +80 (-5, +90) TATCCCTGAGGAATATGTGCTGACCCGGCTGGCAGAGGACCCTGCAGAGCCCAGGTACCGT----- CGAGAGGAGGGCCCGCTTCGTG GCTGACCCGGCTGGCAGACCCGGCTGGCAGAGGACCCTGCAGAGCCCAGGTGCTGACCCGGCTGGCAGAGGACCCTGCAGAGCCCAGGT Kcnj11-/-βCL2 allele 2: +124 (+168, -44) TATCCCTGAGGAATATGTGCTGACCCGGCTGGCAGAGGACCCTGCAGAGCCCAGGTACCGTACTCGAGAGAGGAGGGCCCGCTTCGTG GAAAATGGATATACAAGCTCCCGGGAGCTTTTTGCAAAAGCCTAGGCCTCCAAAAAAGCCTCCCCACTACTTCTGGAATAGCTCAGAGGCAGA GGCGGCCTCGGCCTCTGCATAAATAAAAAAAATTAGTCAGCCATGGGGCGGAGAATGGGCGGAACTGGGCGGAG E KATP channel activity F Insulin secretion Parental -/- -/- 100pA Control Diazoxide Tolbutamide Kcnj11 βCL1 Kcnj11 βCL2 ** ** Parental (MIN6-K8) Kcnj11-/- βCL1 * n.s. n.s. Kcnj11-/- βCL2 n.s. 5 ** content) (% ** Insulin secretion 4 Control (C) 3 Diazoxide (100µM) (D) Glucose (mM) 2.8 2.8 16.7 2.8 2.8 16.7 2.8 2.816.7 100 nM Glibenclamide - + - - + - - + - /pF) Tolbutamide (100µM) (T) Parental Kcnj11-/- βCL1Kcnj11-/- βCL2 nS 2 ( 1 Normalized conductance Normalized 0 CDTCDTCDT Parental Kcnj11-/- βCL1 Kcnj11-/- βCL2 cAMP G Insulin secretion H n.s. 12 ** ** 800 Parental 10 700 Kcnj11-/- βCL1 * ** /ng DNA) 600 8 ** * 500 6 fmol 400 4 * (% content) (% 300 ** * Insulin secretion 2 200 0 100 Glucose (11.1 mM) + + + + + + 0 cAMP content ( cAMP 0 0.1 0.5 1.0 GLP-1 (1 nM) - + - - + - GLP-1 (nM) - - + - - + 2.8 mM G mM 2.8 GIP (1 nM) G mM 2.8 Parental Kcnj11-/- βCL1 5 I PKC activity Phosphoserine- PKC substrates β-actin -/- Parental Kcnj11-/- βCL Parental Kcnj11 βCL Marker 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 (kDa) 1. 2.8 mM Glucose 200 2. 2.8 mM Glucose + 100 nM BIM-1 116 3. 2.8 mM Glucose + 1 nM GLP-1 97.4 4. 2.8 mM Glucose + 1 nM GLP-1 + 100 nM BIM-1 66 5. 2.8 mM Glucose + 5 µM Carbachol 6. 2.8 mM Glucose + 5 µM Carbachol + 100 nM BIM-1 45 BIM-1: Bisindolylmaleimide-1; a selective PKC inhibitor Densitometric analysis 8 J Intracellular calcium actin ratio actin - 6 2.4 2.2 4 380 2 /F 2 340 1.8 ΔF 0 1.6 1 2 3 4 5 6 1 2 3 4 5 6 1.4 ser PKC substrates/β PKC ser Parental Kcnj11-/- βCL1 0 1 2 3 4 P- Time (min) K Gs and Gq protein expression -/- Parental Kcnj11 βCL Parental Kcnj11-/- βCL Marker Marker Parental Kcnj11-/- βCL 1 2 1 2 1 2 1 2 Marker kDa kDa kDa 1 2 1 2 200 200 200 116 116 97.4 116 97.4 97.4 66 66 66 46 β-actin (MW 45 Gαq Gαs 46 46 kDa) (MW 45 (MW 45 31 kDa) kDa) 31 31 21.5 14.5 1.0 1.0 actin ratio actin 0.5 0.5 actin ratio actin Gαq/β- 0.0 Gαs/β- 0.0 1 1 CL CL β β - / - - / - Parental Parental Kcnj11 Kcnj11 6 Oduori et al. Supplementary Figure 3. Expressions of Glp-1r and Gipr (A), adenylyl cyclases (Adcy) (B), and phosphodiesterases (Pde) (C) in pancreatic islets assessed by qPCR (n = 3 for both mouse groups for each gene expression).
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    Figure S1. SYT1 and SYT3 are induced under abiotic stress (related to Figure 1). (A) Phylogenetic clustering of the Arabidopsis protein coding sequence of SYT1, SYT2, SYT3, SYT4, SYT5 and the Homo sapiens E-Syt1. Phylogenetic tree was inferred using the Neighbor-Joining method. The optimal tree with the sum of branch length = 3.26 is shown. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the p-distance method and are in the units of the number of amino acid differences per site. This analysis involved 11 amino acid sequences. All ambiguous positions were removed for each sequence pair (pairwise deletion option). There were a total of 1752 positions in the final dataset. Evolutionary analyses were conducted using MEGA X (Kumar et al., 2018). (B) Gene expression profiles based on RNA-seq analysis of SYT1, SYT2, SYT3, SYT4, and SYT5 in vegetative tissues at different developmental stages from TRAVA database http://travadb.org/). Each dot represent a RNAseq value and the bar represent the median. (C) Gene expression profiles based on RNA-seq analysis of SYT1, SYT3, and SYT5 in eFP-seq Browser after various abiotic stresses (https://bar.utoronto.ca/eFP-Seq_Browser/).https://bar.utoronto.ca/eFP- Seq_Browser/). (D) Heatmap representing the expression responses to abiotic stress of SYT1, SYT3 and SYT5. Expression levels genes are represented as the fold-change relative to the control. Red represents a gene that is induced, and blue represents a gene that is repressed in response to the indicated abiotic stress.
  • Table S2.Up Or Down Regulated Genes in Tcof1 Knockdown Neuroblastoma N1E-115 Cells Involved in Differentbiological Process Anal

    Table S2.Up Or Down Regulated Genes in Tcof1 Knockdown Neuroblastoma N1E-115 Cells Involved in Differentbiological Process Anal

    Table S2.Up or down regulated genes in Tcof1 knockdown neuroblastoma N1E-115 cells involved in differentbiological process analysed by DAVID database Pop Pop Fold Term PValue Genes Bonferroni Benjamini FDR Hits Total Enrichment GO:0044257~cellular protein catabolic 2.77E-10 MKRN1, PPP2R5C, VPRBP, MYLIP, CDC16, ERLEC1, MKRN2, CUL3, 537 13588 1.944851 8.64E-07 8.64E-07 5.02E-07 process ISG15, ATG7, PSENEN, LOC100046898, CDCA3, ANAPC1, ANAPC2, ANAPC5, SOCS3, ENC1, SOCS4, ASB8, DCUN1D1, PSMA6, SIAH1A, TRIM32, RNF138, GM12396, RNF20, USP17L5, FBXO11, RAD23B, NEDD8, UBE2V2, RFFL, CDC GO:0051603~proteolysis involved in 4.52E-10 MKRN1, PPP2R5C, VPRBP, MYLIP, CDC16, ERLEC1, MKRN2, CUL3, 534 13588 1.93519 1.41E-06 7.04E-07 8.18E-07 cellular protein catabolic process ISG15, ATG7, PSENEN, LOC100046898, CDCA3, ANAPC1, ANAPC2, ANAPC5, SOCS3, ENC1, SOCS4, ASB8, DCUN1D1, PSMA6, SIAH1A, TRIM32, RNF138, GM12396, RNF20, USP17L5, FBXO11, RAD23B, NEDD8, UBE2V2, RFFL, CDC GO:0044265~cellular macromolecule 6.09E-10 MKRN1, PPP2R5C, VPRBP, MYLIP, CDC16, ERLEC1, MKRN2, CUL3, 609 13588 1.859332 1.90E-06 6.32E-07 1.10E-06 catabolic process ISG15, RBM8A, ATG7, LOC100046898, PSENEN, CDCA3, ANAPC1, ANAPC2, ANAPC5, SOCS3, ENC1, SOCS4, ASB8, DCUN1D1, PSMA6, SIAH1A, TRIM32, RNF138, GM12396, RNF20, XRN2, USP17L5, FBXO11, RAD23B, UBE2V2, NED GO:0030163~protein catabolic process 1.81E-09 MKRN1, PPP2R5C, VPRBP, MYLIP, CDC16, ERLEC1, MKRN2, CUL3, 556 13588 1.87839 5.64E-06 1.41E-06 3.27E-06 ISG15, ATG7, PSENEN, LOC100046898, CDCA3, ANAPC1, ANAPC2, ANAPC5, SOCS3, ENC1, SOCS4,
  • (12) United States Patent (10) Patent No.: US 7.873,482 B2 Stefanon Et Al

    (12) United States Patent (10) Patent No.: US 7.873,482 B2 Stefanon Et Al

    US007873482B2 (12) United States Patent (10) Patent No.: US 7.873,482 B2 Stefanon et al. (45) Date of Patent: Jan. 18, 2011 (54) DIAGNOSTIC SYSTEM FOR SELECTING 6,358,546 B1 3/2002 Bebiak et al. NUTRITION AND PHARMACOLOGICAL 6,493,641 B1 12/2002 Singh et al. PRODUCTS FOR ANIMALS 6,537,213 B2 3/2003 Dodds (76) Inventors: Bruno Stefanon, via Zilli, 51/A/3, Martignacco (IT) 33035: W. Jean Dodds, 938 Stanford St., Santa Monica, (Continued) CA (US) 90403 FOREIGN PATENT DOCUMENTS (*) Notice: Subject to any disclaimer, the term of this patent is extended or adjusted under 35 WO WO99-67642 A2 12/1999 U.S.C. 154(b) by 158 days. (21)21) Appl. NoNo.: 12/316,8249 (Continued) (65) Prior Publication Data Swanson, et al., “Nutritional Genomics: Implication for Companion Animals'. The American Society for Nutritional Sciences, (2003).J. US 2010/O15301.6 A1 Jun. 17, 2010 Nutr. 133:3033-3040 (18 pages). (51) Int. Cl. (Continued) G06F 9/00 (2006.01) (52) U.S. Cl. ........................................................ 702/19 Primary Examiner—Edward Raymond (58) Field of Classification Search ................... 702/19 (74) Attorney, Agent, or Firm Greenberg Traurig, LLP 702/23, 182–185 See application file for complete search history. (57) ABSTRACT (56) References Cited An analysis of the profile of a non-human animal comprises: U.S. PATENT DOCUMENTS a) providing a genotypic database to the species of the non 3,995,019 A 1 1/1976 Jerome human animal Subject or a selected group of the species; b) 5,691,157 A 1 1/1997 Gong et al.
  • CORVET, CHEVI and HOPS – Multisubunit Tethers of the Endo

    CORVET, CHEVI and HOPS – Multisubunit Tethers of the Endo

    © 2019. Published by The Company of Biologists Ltd | Journal of Cell Science (2019) 132, jcs189134. doi:10.1242/jcs.189134 REVIEW SUBJECT COLLECTION: CELL BIOLOGY AND DISEASE CORVET, CHEVI and HOPS – multisubunit tethers of the endo-lysosomal system in health and disease Jan van der Beek‡, Caspar Jonker*,‡, Reini van der Welle, Nalan Liv and Judith Klumperman§ ABSTRACT contact between two opposing membranes and pull them close Multisubunit tethering complexes (MTCs) are multitasking hubs that together to allow interactions between SNARE proteins (Murray form a link between membrane fusion, organelle motility and signaling. et al., 2016). SNARE assembly then provides additional fusion CORVET, CHEVI and HOPS are MTCs of the endo-lysosomal system. specificity and drives the actual fusion process (Ohya et al., 2009; They regulate the major membrane flows required for endocytosis, Stroupe et al., 2009). In this Review, we focus on the role of tethering lysosome biogenesis, autophagy and phagocytosis. In addition, proteins in endo-lysosomal fusion events. individual subunits control complex-independent transport of specific Tethering proteins can be divided into two main groups: long cargoes and exert functions beyond tethering, such as attachment to coiled-coil proteins (Gillingham and Munro, 2003) and microtubules and SNARE activation. Mutations in CHEVI subunits multisubunit tethering complexes (MTCs). MTCs form a lead to arthrogryposis, renal dysfunction and cholestasis (ARC) heterogenic group of protein complexes that consist of up to ten ∼ syndrome, while defects in CORVET and, particularly, HOPS are subunits resulting in a general length of 50 nm (Brocker et al., associated with neurodegeneration, pigmentation disorders, liver 2012; Chou et al., 2016; Hsu et al., 1998; Lürick et al., 2018; Ren malfunction and various forms of cancer.