Alvogen, Inc. LEVOTHYROXINE SODIUM- levothyroxine sodium ...
Supporting Information - PNAS · For regular IP, cells were lysedinNonidetP-40buffer[50mMTris·Cl,...
6
Supporting Information Zhang et al. 10.1073/pnas.1614467113 SI Materials and Methods Adipose Transplantation. The hair in the abdominal and dorsal regions of mice was shaved before surgery. During surgery, recipient mice were anesthetized under isofluorane vapor [2–5% (vol/vol), 2% O 2 ]. Donor fat pads were freshly isolated from mice eutha- nized by CO 2 inhalation and cervical dislocation and placed into sterile PBS, then immediately loaded in a syringe through the plunger end. The injection was done immediately, to ensure a warm ischemia time of <10 min. iWATs from three 4-wk-old male C57BL/6J donor mice (six iWATs total) were transplanted into one 4-wk-old male recipient mouse. Multiple injections of donor fat pads were made s.c. with 16-gauge needles. Injection volume was kept between 100–150 μL per injection site. The injections were first made in the abdominal region and then, if needed, into the dorsal region. The injection sites were closed with tissue adhesive. Immunohistochemisty and Immunostaining. Samples for routine H&E were immersion-fixed for 48 h in 10% (vol/vol) neutral- buffered formalin and stored briefly in 50% (vol/vol) ethanol, and samples for ORO staining were fixed in methanol-free 4% (vol/vol) paraformaldehyde for 48 h before equilibration in 18% (wt/vol) sucrose. Subsequent paraffin processing and embedding (H&E) and cryoembedding (ORO) were carried out, and sections were cut on a rotary microtome and cryostat, respectively. The resulting sections were stained for routine histopathological evaluation by regressive H&E on a Sakura Finetek DRS-601 robotic staining system using Leica Selectech reagents (hematoxylin 560 and alcoholic eosin Y 515). Foamy and vacuolated histopathologi- cal lesions were differentially determined to consist of lipid accumulations by ORO staining, performed manually accord- ing to established protocols. For immunostaining, 293T cells cultured in chambers were washed with PBS and fixed in freshly made 4% (vol/vol) formal- dehyde in PBS buffer at room temperature for 10 min, then washed again with PBS, treated with PBST [PBS and 0.25% (vol/vol) Triton X-100] for permeabilization, and blocked with PBSA [PBS and 1% (wt/vol) BSA] for 15 min. Cells were incubated with primary antibody diluted in PBSA for 1 h at room temperature, then washed with PBS and incubated with secondary antibody diluted in PBSA for 30 min at room temperature, and finally mounted in mounting medium with DAPI (Life Technologies). Sample Preparation, IP, Ubiquitination, Ub Chain Assay, GST Pull- Down, and Western Blot Analysis. For direct Western blot analy- sis, cells were harvested in 1× NuPAGE LDS sample buffer (Life Technologies) with 2.5% (vol/vol) 2-mercaptoethanol (Sigma- Aldrich). Tissue proteins were isolated from standard TRIzol protocol as pellets and dissolved in denaturing buffer [1% (wt/vol) SDS, 50 mM Tris·Cl, pH 7.5, 0.5 mM EDTA, and 1 mM DTT]. The protein concentration was determined using the Pierce BCA Pro- tein Assay Kit (Thermo Fisher Scientific). For regular IP, cells were lysed in Nonidet P-40 buffer [50 mM Tris·Cl, pH 8.0, 0.1 M NaCl, 10 mM sodium fluoride, 1 mM sodium vanadate, 1% (vol/vol) Nonidet P-40, 10% (vol/vol) glycerol, 1.5 mM EDTA, and Protease Inhibitor Mixture] for 30 min at 4 °C. For IP in the ubiquitination assay, cells were first lysed in denaturing buffer. After incubation for 5 min at 100 °C, the lysates were diluted 10-fold with Nonidet P-40 buffer. After centrifugation, lysates were incubated with indicated antibody-conjugated beads for 2 h at 4 °C. Beads were washed three times with 1 mL of Nonidet P-40 buffer and then boiled in 30 μL of 1× NuPAGE LDS sample buffer with 2.5% (vol/vol) 2- mercaptoethanol. The Ub chain assay was performed with the UbiCREST Deubiquitinase Enzyme Kit (Boston Biochem). Ubiquitinated p85α was purified with anti-FLAG M2 affinity gel (Sigma-Aldrich) un- der the same conditions as for the ubiquitination assay. Purified ubiquitinated p85α beads were mixed with different deubiquiti- nating enzymes at 37 °C for 30 min. The supernatants of reactions were collected for immunoblotting to detect released Ub, and the beads were collected for immunoblotting to detect p85α. GST fusion proteins were produced in Escherichia coli BL21 and purified with glutathione agarose beads (GE Healthcare). GST fusion protein-loaded beads were incubated with 3×FLAG-Kbtbd2 purified from 293T cells in GST pull-down lysis buffer [20 mM Tris·Cl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5% (vol/vol) Nonidet P-40, and PMSF] at 4 °C for 1 h. The beads were washed three times with lysis buffer, followed by Western blot analysis. In a typical Western blot, samples were resolved by NuPAGE 4–12% (wt/vol) Bis-Tris gels (Thermo Fisher Scientific), trans- ferred to NC membranes (Bio-Rad), blotted with the primary antibody at 4 °C overnight and the secondary antibody for 1 h at room temperature, and then visualized by chemiluminescent substrate (Thermo Fisher Scientific). The following primary antibodies were used in this study: mouse anti-HA, anti-FLAG, anti-Cul3 (Sigma-Aldrich), anti-V5 (Invitrogen), anti-K48 link- age (Millipore), rabbit anti-p85α, anti-p85β, anti-p110β, anti–p- Tyr, anti–IRS-1, anti-AKT, anti–p-AKT S473 , anti–β-actin, anti- PPARγ, anti-CEBP/α, anti-FAS, anti-ACC (Cell Signaling Technology), and anti-Kbtbd2 (Abcam). Mass Spectrometry Analysis of Proteins in Adipose Tissues. Proteins in gel bands were reduced and alkylated with DTT and iodoacetamide (Sigma-Aldrich), then digested overnight with trypsin (Pierce). Following solid-phase extraction cleanup with Oasis HLB plates (Waters), the resulting peptides were recon- stituted in 10 μL of 2% (vol/vol) acetonitrile (ACN) and 0.1% trifluoroacetic acid in water. Then 2 μL of this mixture was in- jected and analyzed by LC/MS/MS using an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific) coupled to an Ultimate 3000 RSLC-Nano liquid chromatography system (Dionex). Sam- ples were separated on a 75-μm-i.d., 50-cm Easy Spray column (Thermo Fisher Scientific) packed with 2-μ C18 beads, and eluted with a gradient from 1% to 28% buffer B over 60 min at a flow rate of 250 nL/min. Buffer A contained 2% (vol/vol) ACN and 0.1% formic acid in water, and buffer B contained 80% (vol/vol) ACN, 10% (vol/vol) trifluoroethanol, and 0.08% formic acid in water. The mass spectrometer was operated in positive ion mode with a source voltage of 2.4 kV, capillary temperature of 250 °C, and S-lens RF level at 60.0%. MS scans were acquired at 240,000 resolution, and up to 14 MS/MS spectra were obtained for each full spectrum acquired using collisionally induced dissociation for ions with charge ≥2. Raw MS data files were converted to a peak list format and analyzed using the Central Proteomics Facilities Pipeline (CPFP), version 2.0.3 (42). Peptide identification was per- formed using the Tandem (43) and Open MS Search Algorithm (OMSSA) (44) search engines against the mouse protein da- tabase from Uniprot, with common contaminants and reversed decoy sequences appended (45). Fragment and precursor tolerances of 20 ppm and 0.5 Da were specified, and three missed cleavages were allowed. Carbamidomethylation of Cys was set as a fixed modification, with oxidation of Met set as a variable modification. Zhang et al. www.pnas.org/cgi/content/short/1614467113 1 of 6
Transcript of Supporting Information - PNAS · For regular IP, cells were lysedinNonidetP-40buffer[50mMTris·Cl,...
Supporting InformationZhang et al. 10.1073/pnas.1614467113SI Materials and MethodsAdipose Transplantation. The hair in the abdominal and dorsalregions of mice was shaved before surgery. During surgery, recipientmice were anesthetized under isofluorane vapor [2–5% (vol/vol),2% O2]. Donor fat pads were freshly isolated from mice eutha-nized by CO2 inhalation and cervical dislocation and placed intosterile PBS, then immediately loaded in a syringe through theplunger end. The injection was done immediately, to ensure awarm ischemia time of <10 min. iWATs from three 4-wk-old maleC57BL/6J donor mice (six iWATs total) were transplanted intoone 4-wk-old male recipient mouse. Multiple injections of donorfat pads were made s.c. with 16-gauge needles. Injection volumewas kept between 100–150 μL per injection site. The injections werefirst made in the abdominal region and then, if needed, into thedorsal region. The injection sites were closed with tissue adhesive.
Immunohistochemisty and Immunostaining. Samples for routineH&E were immersion-fixed for 48 h in 10% (vol/vol) neutral-buffered formalin and stored briefly in 50% (vol/vol) ethanol, andsamples for ORO staining were fixed in methanol-free 4% (vol/vol)paraformaldehyde for 48 h before equilibration in 18% (wt/vol)sucrose. Subsequent paraffin processing and embedding (H&E)and cryoembedding (ORO) were carried out, and sections were cuton a rotary microtome and cryostat, respectively. The resultingsections were stained for routine histopathological evaluation byregressive H&E on a Sakura Finetek DRS-601 robotic stainingsystem using Leica Selectech reagents (hematoxylin 560 andalcoholic eosin Y 515). Foamy and vacuolated histopathologi-cal lesions were differentially determined to consist of lipidaccumulations by ORO staining, performed manually accord-ing to established protocols.For immunostaining, 293T cells cultured in chambers were
washed with PBS and fixed in freshly made 4% (vol/vol) formal-dehyde in PBS buffer at room temperature for 10min, then washedagain with PBS, treated with PBST [PBS and 0.25% (vol/vol)Triton X-100] for permeabilization, and blocked with PBSA [PBSand 1% (wt/vol) BSA] for 15 min. Cells were incubated withprimary antibody diluted in PBSA for 1 h at room temperature,then washed with PBS and incubated with secondary antibodydiluted in PBSA for 30 min at room temperature, and finallymounted in mounting medium with DAPI (Life Technologies).
Sample Preparation, IP, Ubiquitination, Ub Chain Assay, GST Pull-Down, and Western Blot Analysis. For direct Western blot analy-sis, cells were harvested in 1× NuPAGE LDS sample buffer (LifeTechnologies) with 2.5% (vol/vol) 2-mercaptoethanol (Sigma-Aldrich). Tissue proteins were isolated from standard TRIzolprotocol as pellets and dissolved in denaturing buffer [1% (wt/vol)SDS, 50 mMTris·Cl, pH 7.5, 0.5 mMEDTA, and 1 mMDTT]. Theprotein concentration was determined using the Pierce BCA Pro-tein Assay Kit (Thermo Fisher Scientific). For regular IP, cells werelysed in Nonidet P-40 buffer [50 mM Tris·Cl, pH 8.0, 0.1 M NaCl,10 mM sodium fluoride, 1 mM sodium vanadate, 1% (vol/vol)Nonidet P-40, 10% (vol/vol) glycerol, 1.5 mM EDTA, and ProteaseInhibitor Mixture] for 30 min at 4 °C. For IP in the ubiquitinationassay, cells were first lysed in denaturing buffer. After incubation for5 min at 100 °C, the lysates were diluted 10-fold with Nonidet P-40buffer. After centrifugation, lysates were incubated with indicatedantibody-conjugated beads for 2 h at 4 °C. Beads were washed threetimes with 1 mL of Nonidet P-40 buffer and then boiled in 30 μLof 1× NuPAGE LDS sample buffer with 2.5% (vol/vol) 2-mercaptoethanol.
The Ub chain assay was performed with the UbiCRESTDeubiquitinase Enzyme Kit (Boston Biochem). Ubiquitinated p85αwas purified with anti-FLAG M2 affinity gel (Sigma-Aldrich) un-der the same conditions as for the ubiquitination assay. Purifiedubiquitinated p85α beads were mixed with different deubiquiti-nating enzymes at 37 °C for 30 min. The supernatants of reactionswere collected for immunoblotting to detect released Ub, and thebeads were collected for immunoblotting to detect p85α.GST fusion proteins were produced in Escherichia coli BL21
and purified with glutathione agarose beads (GE Healthcare).GST fusion protein-loaded beads were incubated with3×FLAG-Kbtbd2 purified from 293T cells in GST pull-downlysis buffer [20 mM Tris·Cl, pH 8.0, 200 mM NaCl, 1 mM EDTA,0.5% (vol/vol) Nonidet P-40, and PMSF] at 4 °C for 1 h. Thebeads were washed three times with lysis buffer, followed byWestern blot analysis.In a typical Western blot, samples were resolved by NuPAGE
4–12% (wt/vol) Bis-Tris gels (Thermo Fisher Scientific), trans-ferred to NC membranes (Bio-Rad), blotted with the primaryantibody at 4 °C overnight and the secondary antibody for 1 h atroom temperature, and then visualized by chemiluminescentsubstrate (Thermo Fisher Scientific). The following primaryantibodies were used in this study: mouse anti-HA, anti-FLAG,anti-Cul3 (Sigma-Aldrich), anti-V5 (Invitrogen), anti-K48 link-age (Millipore), rabbit anti-p85α, anti-p85β, anti-p110β, anti–p-Tyr, anti–IRS-1, anti-AKT, anti–p-AKTS473, anti–β-actin, anti-PPARγ, anti-CEBP/α, anti-FAS, anti-ACC (Cell SignalingTechnology), and anti-Kbtbd2 (Abcam).
Mass Spectrometry Analysis of Proteins in Adipose Tissues. Proteinsin gel bands were reduced and alkylated with DTT andiodoacetamide (Sigma-Aldrich), then digested overnight withtrypsin (Pierce). Following solid-phase extraction cleanup withOasis HLB plates (Waters), the resulting peptides were recon-stituted in 10 μL of 2% (vol/vol) acetonitrile (ACN) and 0.1%trifluoroacetic acid in water. Then 2 μL of this mixture was in-jected and analyzed by LC/MS/MS using an Orbitrap Elite massspectrometer (Thermo Fisher Scientific) coupled to an Ultimate3000 RSLC-Nano liquid chromatography system (Dionex). Sam-ples were separated on a 75-μm-i.d., 50-cm Easy Spray column(Thermo Fisher Scientific) packed with 2-μ C18 beads, and elutedwith a gradient from 1% to 28% buffer B over 60 min at a flow rateof 250 nL/min. Buffer A contained 2% (vol/vol) ACN and 0.1%formic acid in water, and buffer B contained 80% (vol/vol) ACN,10% (vol/vol) trifluoroethanol, and 0.08% formic acid in water.The mass spectrometer was operated in positive ion mode with asource voltage of 2.4 kV, capillary temperature of 250 °C, andS-lens RF level at 60.0%. MS scans were acquired at 240,000resolution, and up to 14MS/MS spectra were obtained for each fullspectrum acquired using collisionally induced dissociation for ionswith charge ≥2.Raw MS data files were converted to a peak list format
and analyzed using the Central Proteomics Facilities Pipeline(CPFP), version 2.0.3 (42). Peptide identification was per-formed using the Tandem (43) and Open MS Search Algorithm(OMSSA) (44) search engines against the mouse protein da-tabase from Uniprot, with common contaminants and reverseddecoy sequences appended (45). Fragment and precursortolerances of 20 ppm and 0.5 Da were specified, and threemissed cleavages were allowed. Carbamidomethylation of Cyswas set as a fixed modification, with oxidation of Met set as avariable modification.
Zhang et al. www.pnas.org/cgi/content/short/1614467113 1 of 6
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Fig. S1. The teeny phenotype. (A) Photographs of homozygous tny mice and WT littermates at different ages after birth. (B) Growth curve of male ho-mozygous tnymice (n = 3) and WT littermates (n = 3) from 4 wk of age to 29 wk of age. (C) Serum IGF1 in 8-wk-old male mice. (D and E) Food (D) and water (E)intake of 8-wk-old male mice. (F) Photographs of cages with 8-wk-old male homozygous tnymice (n = 4) or WT littermates (n = 4) at 2 d after cage change. Thetny/tny cage was wetted with urine (arrow). (G and H) Blood glucose (G) and serum insulin (H) were measured in male homozygous tny mice (n = 3) and WTlittermates (n = 3) at weekly intervals, each time after a 6-h fast, beginning at 4 wk of age. (I) HbA1c in the blood of 8-wk-old male mice. (J) Immunoblots ofliver and adipose tissue lysates from 4-wk-old male homozygous tny/tny mice and WT littermates, with or without insulin injection. Insulin or saline was in-jected i.p. at a dose of 1.5 U/kg body weight. Tissues were removed for analysis at 30 min postinjection. (K and L) Body weight (K) and blood glucose levels (L)of male homozygous tny, heterozygous tny, and WT mice at 8 wk of age. Glucose was measured after a 6-h fast. In C–E, H, K, and L, data points representindividual mice. P values were determined by Student’s t test.
Zhang et al. www.pnas.org/cgi/content/short/1614467113 2 of 6
A B
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Fig. S2. Adipose transplantation of tny mice. (A) Representative photograph of a Kbtbd2−/− mouse at 12 wk after transplantation of adipose tissue from a WTmouse (Kbtbd2−/− AT+). Skin was dissected to show the adipose tissue grafts (white arrowheads). (B) Enlarged view of an adipose tissue graft showing theblood vessels (arrowheads) supplying the graft. (C) H&E staining of a section of grafted adipose tissue. Red blood cells are visible (arrowheads) in the bloodvessels.
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Fig. S3. Subcellular location of the KBTBD2 protein and expression profile of Kbtbd2 transcript. (A) 293T cells expressing 3×FLAG-tagged KBTBD2 proteinwere immunostained with FLAG (green) antibody, phalloidin (red) to visualize F-actin, and DAPI (blue) to visualize nuclei. (B) Kbtbd2 transcript levels nor-malized to Actb mRNA in different tissues of male C57BL/6J mice at 8 wk of age (n = 3).
Zhang et al. www.pnas.org/cgi/content/short/1614467113 3 of 6
Fig. S4. KBTBD2 regulates p85α in differentiated human adipocytes. (A) Immunoblots of lysates of differentiated human adipocytes expressing control shRNAor two different Kbtbd2 shRNAs. (B) Immunoblot analysis of immunoprecipitates (Top and Middle) or lysates (Bottom) of differentiated human adipocytesexpressing the indicated tagged proteins. (C) Immunoblots of lysates of differentiated human adipocytes expressing control shRNA or two different Kbtbd2shRNAs with or without insulin treatment.
Fig. S5. KBTBD2 deficiency impairs the metabolic function of MEF-derived adipocytes. MEF cells isolated from two WT embryos or Kbtbd2−/− embryos weredifferentiated in vitro for 10 d to generate MEF-derived adipocytes. Immunoblots of lysates of original MEFs or MEF-derived adipocytes are shown.
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Fig. S6. Phenotypes of Lepob/ob, Leprdb/db, and Mc4r−/− mice. (A–E) Male Lepob/+ (ob/+) or Lepob/ob (ob/ob) littermates were maintained on the control diet (n = 3)or high-fat diet (n = 3) for 8 wk, beginning at 6 wk of age. Experiments were conducted at 14 wk of age. (F–J) Male Leprdb/+ (db/+; n = 3) and Leprdb/db (db/db; n = 3)littermates were maintained on standard chow from birth. Experiments were conducted at 16 wk of age. (K–O) MaleMc4r−/− mice (n = 3) and WT mice (+/+; n = 3)were maintained on standard chow from birth. Experiments were conducted at 9 wk of age. (A, F, and K) Body weight. (B, G, and L) Normalized fat weight. (C, H,and M) Blood glucose levels. (D, I, and N) Serum insulin levels. Glucose and insulin were measured after a 6-h fast. (E, J, and O) Insulin tolerance test. Blood glucosewas measured at indicated times after i.p. insulin injection. Baseline blood glucose levels (0 min): control diet [ob/+, 218 ± 20 mg/dL; ob/ob, 361 ± 6 mg/dL]; high-fatdiet [ob/+, 325 ± 10 mg/dL; ob/ob, 539 ± 12 mg/dL; db/+, 186 ± 11 mg/dL; db/db, 406 ± 28 mg/dL;Mc4r+/+, 176 ± 17 mg/dL;Mc4r−/−, 290 ± 10 mg/dL]. In A–D, F–I, andK–N, data points represent individual mice. P values were determined by Student’s t test.
Table S1. Non-Mendelian birth ratio of tny heterozygousbreedings
Genotype Pup numbers Percentage
+/+ 131 32.5%tny/+ 236 58.6%tny/tny 36 8.9%
Zhang et al. www.pnas.org/cgi/content/short/1614467113 5 of 6
Table S2. Percent pairwise identity of KBTBD2 amino acid sequences
SpeciesMus
musculusHomosapiens
Pantroglodytes
Macacamulatta
Canislupus
Bostaurus
Rattusnorvegicus
Gallusgallus
Xenopustropicalis Danio rerio
Mus musculus 100.0Homo sapiens 98.6 100.0Pan troglodytes 98.4 99.8 100.0Macaca mulatta 98.6 100 99.8 100.0Canis lupus 99.0 99.4 99.2 99.4 100.0Bos taurus 99.4 98.9 98.7 98.9 99.4 100.0Rattus norvegicus 99.7 98.7 98.6 98.7 99.0 99.4 100.0Gallus gallus 93.7 94.4 94.2 94.4 94.7 93.6 94.1 100.0Xenopus tropicalis 84.7 85.0 84.9 85.0 85.4 82.3 85.0 86.2 100.0Danio rerio 79.3 79.0 78.8 79.0 79.3 78.5 79.6 80.1 74.4 100.0
Table S3. Proteins identified by mass spectrometry that were increased by more than fivefoldin multiple Kbtbd2−/− adipose tissue samples relative to WT samples
Protein Description PSMs Peptide seqs % Coverage eWAT KO/WT iWAT KO/WT
P26450 p85α 36 23 34.80 41.293 24.196Q00PI9 Hnrnpul2 25 11 22.10 16.084 5.948Q6XLQ8 Calu 23 7 34.30 5.313 49.732P01898 H2-Q10 14 12 41.80 6.939 20.329Q60749 Khdrbs1 7 4 7.20 11.495 25.291Q9R0A0 Pex14 6 3 8.20 47.966 25.154
Other Supporting Information Files
Dataset S1 (XLSX)
Zhang et al. www.pnas.org/cgi/content/short/1614467113 6 of 6
![Supporting Information · 2019. 3. 13. · Figure S1. ITC of YL (0.588 mM or 0.592 mM) titrated into CB[8] (0.053 mM). 10 mM sodium phosphate buffer (pH 7.0)., 298.15 K, iTC200, 25](https://static.fdocuments.in/doc/165x107/6126903b336f2866635c7320/supporting-2019-3-13-figure-s1-itc-of-yl-0588-mm-or-0592-mm-titrated-into.jpg)
