J. Cell Sci. : doi:.4/jcs.59: Supplementary information E9. A Arl8b /- Arl8b -/- Arl8b Arl8b non-specific band Gapdh Tbp E7.5 HE Inset B D Control al am hf C E Arl8b -/- al am hf E8.5 F low middle high G genotype electron density high middle low Arl8b /... Arl8b -/- 6.7.5 5.9 Fig. S. Arl8b / mutant phenotypes. (A) RT-PCR analysis of Arl8b mrna in E9. embryos. (B E) HE-stained transverse cryosections of E7.5 embryos. The insets in (B) and (C) are magnified in (D) and (E), respectively. Bars, μm. (F) Examples of high-, middle-, and low-electron density vesicles in the Arl8b / VYSE. Vesicles were classified into three groups on the basis of their electron densities: vesicles with suprathreshold intensity across their whole area were classified as high-density, those with subthreshold intensity across their whole area as low-density, and others as middle-density. Bar, 5. μm. (G) The numbers of vesicles per 5 cells were scored for wild-type and Arl8b / VYSE (n = ).
J. Cell Sci. : doi:.4/jcs.59: Supplementary information Visceral Yolk Sac (E9.5) 8 6 4 Glycine Ala Alanine Isoleucine Ile 5 5 5 5 6 BCAA Val Valine 5 5 4 4 Serine Threonine 5 Ser Thr 4 Leucine 5 5 5 5 Lysine Arginine His Histidine Methionine 5 8 8 4 6 6 4 4 Tyr Phe Tyrosine Phenylalanine Tryptophan 4 5 Trp BAA AAA 8 6 Pro Proline 4 5 5 5 5 5 Gln Glutamine Glutamic acid Asn Asparagine Aspartic acid 5 5 8 4 5 6 4 : Arl8b flox/ or Arl8b flox/flox : Ttr-Cre; Arl8b flox/ : Ttr-Cre; Arl8b flox/flox Fig. S. Alterations of amino acid level in the VYS of the Ttr-Cre;Arl8b flox/flox embryo. Relative amounts of amino acids in the VYS of the E9.5 embryo. BCAA, BAA, and AAA represent branched-chain amino acids, basic amino acids, and aromatic amino acids, respectively. The scale of the vertical axis in each panel is given in arbitrary units. P<.5, P<., P<..
J. Cell Sci. : doi:.4/jcs.59: Supplementary information Embryo Glycolysis 5 5 Glucose Glucose-6-phosphate 5 glucose 6-phosphate dehydrogenase Pentose phosphate pathway 6-Phosphogluconoδ-lactone 6-Phosphogluconate 8 6 4 Ribulose 5-phosphate 5 ribulose phosphate -epimerase ribose 5-phosphate isomerase phosphoglucose isomerase Fructose-6-phosphate 5 transketolase Xylulose 5-phosphate Ribose 5-phosphate 5 Fructose 6-phosphate Glyceraldehyde -phosphate fructose,6-bisphosphatase phosphofructokinase- Fructose-,6-diphosphate 5 5 5 5 Erythrose 4-phosphate 8 6 4 Sedoheptulose 7-phosphate Dihydroxyacetone phosphate (DHAP) TPI Aldolase, Diphospho glycerate 4 5 5 5 DHAPGA--P 5 5 5 : Arl8b flox/ or Arl8b flox/flox : Ttr-Cre; Arl8b flox/ : Ttr-Cre; Arl8b flox/flox 6 4 Glyceraldehyde -phosphate (GA--P) Gapdh, Di-phospho glycerate Glycine PGK -Phosphoglycerate 5 5 -Phospho-D-glycerate Fig. S. Metabolite levels of glycolysis and pentose phosphate pathway in the embryo proper. Relative amounts of metabolites of glycolysis and pentose phosphate pathway in the embryo proper of the E9.5 embryo. The scale of the vertical axis in each panel is given in arbitrary units. P<.5. 5 4 PGM Lactate ENO Phosphoenolpyruvate 5 5 9 8 7 6 5 4 LDH PK Pyruvate TCA Cycle
J. Cell Sci. : doi:.4/jcs.59: Supplementary information Embryo Ketone-Bodies β-hydroxybutyrate 5 5 5 9 8 7 6 5 4 Malate Mevalonate Acetoacetate Acetyl-CoA Oxaloacetate 6 4 CoASH 9 8 7 6 5 4 Glucose Pyruvate Citrate 5 4 Pantothenate cis-aconitate 4 Fumarate 5 5 TCA cycle 4 Iso-citrate 8 6 4 Succinate : Arl8b flox/ or Arl8b flox/flox : Ttr-Cre; Arl8b flox/ : Ttr-Cre; Arl8b flox/flox Succinyl-CoA α-ketoglutarate 5 5 Glu AAs N-Acetyl-L-glutamic acid Fig. S4. Metabolite levels of TCA cycle in the embryo proper. Relative amounts of metabolites of TCA cycle in the embryo proper of the E9.5 embryo. The scale of the vertical axis in each panel is given in arbitrary units. P<.5. 5 5 5
J. Cell Sci. : doi:.4/jcs.59: Supplementary information Visceral Yolk Sac Glycolysis 6 4 Glucose Glucose-6-phosphate 4 glucose 6-phosphate dehydrogenase Pentose phosphate pathway 6-Phosphogluconoδ-lactone 6-Phosphogluconate 5 5 Ribulose 5-phosphate ribulose phosphate -epimerase ribose 5-phosphate isomerase phosphoglucose isomerase Fructose-6-phosphate 4 transketolase Xylulose 5-phosphate Ribose 5-phosphate Fructose 6-phosphate Glyceraldehyde -phosphate fructose,6-bisphosphatase Dihydroxyacetone phosphate (DHAP) phosphofructokinase- Fructose-,6-diphosphate 5, Di-phospho glycerate PGK, Diphospho glycerate -Phosphoglycerate 8 6 4 5 5 5 TPI : Arl8b flox/ or Arl8b flox/flox : Ttr-Cre; Arl8b flox/ : Ttr-Cre; Arl8b flox/flox Aldolase DHAPGA--P 8 6 4 Glyceraldehyde -phosphate (GA--P) Glycine Gapdh 5 5 8 6 4 Erythrose 4-phosphate -Phospho-D-glycerate Fig. S5. Metabolite levels of glycolysis and pentose phosphate pathway in the VYS. Relative amounts of metabolites of glycolysis and pentose phosphate pathway in the VYS of the E9.5 embryo. The scale of the vertical axis in each panel is given in arbitrary units. P<.5, P<.. 8 6 4 PGM Lactate 8 6 4 Sedoheptulose 7-phosphate ENO Phosphoenolpyruvate 5 5 5 5 LDH PK Pyruvate TCA Cycle
J. Cell Sci. : doi:.4/jcs.59: Supplementary information Visceral Yolk Sac Ketone-Bodies β-hydroxybutyrate 5 4 Mevalonate Acetoacetate Acetyl-CoA Glucose Pyruvate 5 5 CoASH 8 6 4 Pantothenate Oxaloacetate 5 Malate 8 6 4 Citrate 5 5 cis-aconitate 8 6 4 Fumarate TCA cycle 5 4 Iso-citrate 5 5 Succinate : Arl8b flox/ or Arl8b flox/flox : Ttr-Cre; Arl8b flox/ : Ttr-Cre; Arl8b flox/flox Succinyl-CoA α-ketoglutarate 5 5 Glu AAs N-Acetyl-L-glutamic acid 5 Fig. S6. Metabolite levels of TCA cycle in the VYS. Relative amounts of metabolites of TCA cycle in the VYS of the E9.5 embryo. The scale of the vertical axis in each panel is given in arbitrary units. P<.5. 5
J. Cell Sci. : doi:.4/jcs.59: Supplementary information Table S. Primers used for construction of a conditional knockout targeting vector. Short arm forward 5 - CCA GCT GGG GCT CGA ATA AGC AGC CCT GTC CCA GTC - Short arm reverse 5 - CGG CCG CCA CCT CGA GAA TTC TCA ATT TGT CCT CTT TTT GGC - Middle arm forward Middle arm reverse Long arm forward 5 - ACG AAG TTA TGT CGA GTG CAT TTT ATT AGC CCT ATT AG - 5 - GGG GGA TGT GGT CGA TCA GAA CTC TAA TTC TAG TGC - 5 - TAT GGA TCC GCG GCC TCT CCG GCC TTG CTT CCT GTG - Long arm reverse 5 - ATG GCC ATA GCG GCC GCA AGC AGG CAG GTC TAG AGT TG - Table S. Primers used for RT-PCR analysis. Arl8b forward 5 - GTG GGG GGG GTA GGG - Arl8b reverse 5 - GTG ACG TTG CCT TTA GTT ACT TTC C - Gapdh forward 5 - CAT GGC CTT CCG TGT TCC TA - Gapdh reverse 5 - GCG GCA CGT CAG ATC CA - Tbp forward 5 - ACC CTT CAC CAA TGA CTC CTA TG - Tbp reverse 5 - TGA CTG CAG CAA ATC GCT TGG -
J. Cell Sci. : doi:.4/jcs.59: Supplementary information Table S. The working dilutions of antibodies used in this study. Antibodies Manufacturers or Ref. Clone or Cat.# Dilution or concentration anti-lamp DSHB D4B /5 for IF anti-albumin Abcam ab658 / for IB anti-cathepsin L R&D AF55. µg/ml for IB anti-actin Merck Millipore MAB5 /5 for IB anti-arl8 Okai et al., 4 N/A /5 for IF, / for IB Anti-Gapdh Merck Millipore MAB74 / for IB IF: Immunofluorescence staining, IB: Immunoblotting, N/A: not applicable Table S4. MRM conditions for each amino acid. Compounds Q m/z Q m/z Collision Energy L-Met 8 56. - Serine 6.5 4.5-4 Asparagine. 87.5 - Aspartic acid 4 74.5-5 Glycine 75.9.5 - Glutamine 47. 84.5-8 Threonine.5 56.5-7 Cysteine.5 59-4 Alanine 9.5 44. -5 Glutamic acid 48.5.5 - Lysine 47. 84. -8 Histidine 55.9. -5 Proline 6. 7.5-5 Arginine 75. 7. - Valine 8. 7.5-5 Methionine 49.9 56. -8 Tyrosine 8. 6. -5 Isoleucine. 86. - Leucine. 86.5 - Phenylalanine 66.. -5 Tryptophan 5. 88.5 -