Journal: Cell Reports

Article Title: IgG targeting distinct seasonal coronavirus- conserved SARS-CoV-2 spike subdomains correlates with differential COVID-19 disease outcomes

doi: 10.1016/j.celrep.2022.110904

Figure Lengend Snippet:

Article Snippet: Human SARS coronavirus (SARS-CoV) Spike glycoprotein Gene ORF cDNA clone expression plasmid (Codon Optimized), C-Flag tag , SinoBiological , VG40150-CF.

Techniques: Virus, Recombinant, Cell Culture, Enzyme-linked Immunosorbent Assay, In Vitro, Transfection, Luciferase, Lysis, Expressing, Plasmid Preparation, Software, Sequencing

Journal: Developmental cell

Article Title: PI5P4Ks drive metabolic homeostasis through peroxisome-mitochondria interplay

doi: 10.1016/j.devcel.2021.04.019

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: This Paper (Primer-BLAST) Table S6 : Mouse qRT-PCR primers A full list of qPCR primers C. elegans synthesized is included in Table S8 Rooney et al., 2015 Table S8 : C.elegans qRT-PCR primers Recombinant DNA pBabe-Zeo-LT-ST A gift from Jorge Moscat N/A pCL-Eco Naviaux et al., 1996 Addgene plasmid Cat # 12371 pCL-Ampho Novus Biologicals Cat# NBP2–29541 pVSV-G A gift from Sammie Jeffrey N/A psPax2 Addgene Cat# 12260 pCF806_Reci A gift from Christof Fellman N/A pCF806_shRenilla A gift from Christof Fellman N/A pCF806_shMulti (a,b) This paper N/A rtTA3 Fellmann et al., 2013 N/A pBabe-3xHA PIP4K2A (WT) This paper N/A pBabe-3xHA PIP4K2A D359N (Kinase-dead) This paper N/A pBabe-3xHA PIP4K2B (WT) This paper N/A RT3REVIN Fellmann et al., 2013 N/A pX330-U6-Chimeric_BB-CBh-hSpCas9 Cong et al., 2013 Addgene plasmid Cat # 42230 Software and algorithms SoftGenetics GeneMarker® software N/A STARv2.3.0e N/A GENCODE v19 N/A STAR (Version 2.4.2) aligner N/A MultiQuant Software (version 3.0.3, Sciex technologies) N/A Image J N/A GraphPad N/A Open in a separate window KEY RESOURCES TABLE PI-4,5-P2 generated at peroxisomes by PI5P4Ks is essential for lipid trafficking Loss of PI5P4K activity leads to peroxisomal dysfunction and accumulation of VLCFAs PI5P4K inhibition impairs mitochondrial function downstream of peroxisomes Therapeutic rationale for targeting PI5P4Ks for metabolically vulnerable diseases

Techniques: Purification, Virus, Plasmid Preparation, Recombinant, Live Cell Imaging, SYBR Green Assay, Bicinchoninic Acid Protein Assay, Reverse Transcription, Quantitation Assay, RNA Sequencing, Sequencing, shRNA, Clone Assay, Synthesized, Software

Journal: EMBO Molecular Medicine

Article Title: An intrinsic mechanism of metabolic tuning promotes cardiac resilience to stress

doi: 10.1038/s44321-024-00132-z

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Signals were detected by the chemiluminescent reagent LiteAblot (Euroclone) at the Chemidoc Touch Imaging System (Bio-Rad).

Techniques: Recombinant, Membrane, Control, Sequencing, BIA-KA, Cell Culture, In Situ, Reverse Transcription, Gene Expression, Enzyme-linked Immunosorbent Assay, ATP Bioluminescent Assay, Electron Microscopy, Flow Cytometry, Software, Imaging, Gas Chromatography, High Performance Liquid Chromatography, Microscopy, Real-time Polymerase Chain Reaction

Journal: Bio-protocol

Article Title: Detection and Differentiation of Multiple Viral RNAs Using Branched DNA FISH Coupled to Confocal Microscopy and Flow Cytometry

doi: 10.21769/BioProtoc.3058

Figure Lengend Snippet: Huh7.5.1 cells were infected with JFH1-CA, JFH1-WT or co-infected at multiplicities of infection equal to one virus per cell with each virus. Infected cells were incubated for 72 h before labeling viral RNAs using PrimeFlow. Analysis and compensation was performed using FlowJo.

Article Snippet: Pipette tips (with or without filter tips) Micro slides (VWR, catalog number: 48311-702) Micro cover glass (VWR, catalog number: 48380-046) GenePulser cuvettes, 4 mm (Bio-Rad Laboratories, catalog number: 1652088) BD FACS tubes (BD Falcon, catalog number: 352054) 12-well cell culture dish ( e.g. , Corning, Costar, catalog number: 3513) 10 cm tissue culture dish ( e.g. , Corning, catalog number: 430167) T150 tissue culture flask ( e.g. , Corning, catalog number: 430825) 15 ml conical centrifuge tube ( e.g. , AccuFlow, catalog number: EK-4020) 500 ml Rapid-Flow Filter Unit, 0.2 μm (Thermo Fisher Scientific, catalog number: 566-0020) Huh7.5.1 cells (Gift from Dr. Michael Gale Jr., University of Washington) PrimeFlow™ RNA Assay Kit (Thermo Fisher Scientific, catalog number: 88-18005-210) contains: Flow Cytometry Staining Buffer Fixation Buffer 1 Permeabilization Buffer with RNase Inhibitors Fixation Buffer 2 Wash Buffer Target Probe Diluent PreAmp Mix Amp Mix Label Probe Diluent 100x Label Probes ViewRNA® Cell Plus Assay Kit (Thermo Fisher Scientific, catalog number: 88-19000) contains: Fixation/Permeabilization Buffer Blocking/Antibody Diluent Fixative Probe Set Diluent Amplifier Diluent along with Pre-Amplifiers and Amplifiers Label Probe Diluent and Label Probes Wash Buffer PBS DAPI Target Probes (Thermo Fisher Scientific) Wild-type JFH1 (VF1-14301) Codon altered JFH1 (VF4-6000723) Permafluor Mounting Reagent (Thermo Fisher Scientific, catalog number: TA-030-FM) 0.05% Trypsin-EDTA (Thermo Fisher Scientific, Gibco, catalog number: 25300-054) Xbal and CutSmart Buffer (New England Biolabs, catalog number: R0145L) MEGAscript T7 Kit (Thermo Fisher Scientific, Invitrogen, catalog number: AMB1334-5) Trizol® Reagent (Thermo Fisher Scientific, Ambion, catalog number: 15596018) QIAquick PCR Purification Kit (QIAGEN, catalog number: 28106) Human AB Serum (Omega, catalog number: HS-20) Pen/Strep (Thermo Fisher Scientific, catalog number: 15140-122) Glutamax (Thermo Fisher Scientific, catalog number: 35050-061) Non-essential amino acids (Thermo Fisher Scientific, catalog number: 11140-050) DMEM (GE Healthcare, Hyclone, catalog number: SH30243.01) Fetal bovine serum (Omega, catalog number: FB-22) KCl CaCl 2 K 2 HPO 4 HEPES EDTA MgCl 2 Human serum media (see Recipes ) 10% FBS media (see Recipes ) CytoMix (see Recipes )

Techniques: Infection, Incubation, Labeling

Journal: Bio-protocol

Article Title: Detection and Differentiation of Multiple Viral RNAs Using Branched DNA FISH Coupled to Confocal Microscopy and Flow Cytometry

doi: 10.21769/BioProtoc.3058

Figure Lengend Snippet: Huh7.5.1 cells were co-infected with JFH1-WT and JFH1-CA on Micro Cover Glass for 72 h. Cells were stained for HCV core protein and both viral RNAs using the ViewRNA Cell Plus Assay. Quantification of colocalization was performed using Volocity software. Scale bars are 2.5 μm in length.

Article Snippet: Pipette tips (with or without filter tips) Micro slides (VWR, catalog number: 48311-702) Micro cover glass (VWR, catalog number: 48380-046) GenePulser cuvettes, 4 mm (Bio-Rad Laboratories, catalog number: 1652088) BD FACS tubes (BD Falcon, catalog number: 352054) 12-well cell culture dish ( e.g. , Corning, Costar, catalog number: 3513) 10 cm tissue culture dish ( e.g. , Corning, catalog number: 430167) T150 tissue culture flask ( e.g. , Corning, catalog number: 430825) 15 ml conical centrifuge tube ( e.g. , AccuFlow, catalog number: EK-4020) 500 ml Rapid-Flow Filter Unit, 0.2 μm (Thermo Fisher Scientific, catalog number: 566-0020) Huh7.5.1 cells (Gift from Dr. Michael Gale Jr., University of Washington) PrimeFlow™ RNA Assay Kit (Thermo Fisher Scientific, catalog number: 88-18005-210) contains: Flow Cytometry Staining Buffer Fixation Buffer 1 Permeabilization Buffer with RNase Inhibitors Fixation Buffer 2 Wash Buffer Target Probe Diluent PreAmp Mix Amp Mix Label Probe Diluent 100x Label Probes ViewRNA® Cell Plus Assay Kit (Thermo Fisher Scientific, catalog number: 88-19000) contains: Fixation/Permeabilization Buffer Blocking/Antibody Diluent Fixative Probe Set Diluent Amplifier Diluent along with Pre-Amplifiers and Amplifiers Label Probe Diluent and Label Probes Wash Buffer PBS DAPI Target Probes (Thermo Fisher Scientific) Wild-type JFH1 (VF1-14301) Codon altered JFH1 (VF4-6000723) Permafluor Mounting Reagent (Thermo Fisher Scientific, catalog number: TA-030-FM) 0.05% Trypsin-EDTA (Thermo Fisher Scientific, Gibco, catalog number: 25300-054) Xbal and CutSmart Buffer (New England Biolabs, catalog number: R0145L) MEGAscript T7 Kit (Thermo Fisher Scientific, Invitrogen, catalog number: AMB1334-5) Trizol® Reagent (Thermo Fisher Scientific, Ambion, catalog number: 15596018) QIAquick PCR Purification Kit (QIAGEN, catalog number: 28106) Human AB Serum (Omega, catalog number: HS-20) Pen/Strep (Thermo Fisher Scientific, catalog number: 15140-122) Glutamax (Thermo Fisher Scientific, catalog number: 35050-061) Non-essential amino acids (Thermo Fisher Scientific, catalog number: 11140-050) DMEM (GE Healthcare, Hyclone, catalog number: SH30243.01) Fetal bovine serum (Omega, catalog number: FB-22) KCl CaCl 2 K 2 HPO 4 HEPES EDTA MgCl 2 Human serum media (see Recipes ) 10% FBS media (see Recipes ) CytoMix (see Recipes )

Techniques: Infection, Staining, Software

Journal: eLife

Article Title: Syntaxin 17 recruitment to mature autophagosomes is temporally regulated by PI4P accumulation

doi: 10.7554/eLife.92189

Figure Lengend Snippet: ( A ) Schematic representation of the structures of STX17 and its C-terminal variants. The positively (orange) and negatively (blue) charged residues are shown. Alanine substitutions are shown in green. TMH, transmembrane helix; CTR, C-terminal region. ( B ) Schematic representation of the localization of ATG5, LC3B, and STX17 during autophagosome formation and maturation. ( C–E ) Mouse embryonic fibroblasts (MEFs) stably expressing mRuby3-LC3B and GFP–STX17TM (containing the two transmembrane helices and the C-terminal region) or its mutants were cultured in starvation medium for 1 hr. Quantification of GFP–STX17TM intensity of mRuby3–LC3B-positive ring-like structures (n>30) are shown in the graphs. In box plots, solid horizontal lines indicate medians, boxes indicate the interquartile ranges (25th to 75th percentiles), whiskers indicate the 5th to 95th percentiles, and dots represent outliers. Differences were statistically analyzed by Welch’s t -test ( C ) or one-way ANOVA followed by Dunnett’s multiple comparison test ( D and E ). Experiments were performed three times independently. Scale bars, 10 μm (main), 1 μm (inset) ( C, D, and E ). Figure 1—source data 1. Data used for graphs presented in and .

Article Snippet: First, cDNAs encoding human STX17 , D. melanogaster STX17 (NM_079202), C. elegans STX17 (NM_059941), rat LC3B , and mouse ATG5 (NM_053069) were inserted into pMRXIP (harboring a puromycin-resistant marker) ( ; ), pMRXIZ (harboring a zeocin-resistant marker) , and pMRXIB (harboring a blasticidin-resistant marker) ( ) together with enhanced GFP or mRuby3 (codon-optimized, Addgene #74252).

Techniques: Stable Transfection, Expressing, Cell Culture, Comparison

Journal: eLife

Article Title: Syntaxin 17 recruitment to mature autophagosomes is temporally regulated by PI4P accumulation

doi: 10.7554/eLife.92189

Figure Lengend Snippet: ( A ) Multiple sequence alignment of STX17 proteins from Homo sapiens (Hs), Mus musculus , Danio rerio , Ciona intestinalis , Drosophila melanogaster (Dm), and Caenorhabditis elegans (Ce). Identical residues between more than two species are indicated with gray boxes. Domains of STX17 are indicated with different colors: green, the Habc domain; magenta, the SNARE domain; blue, transmembrane helix (TMH); orange, a linker between the TMDs; and purple, the C-terminal region. ( B ) Mouse embryonic fibroblasts (MEFs) stably expressing mRuby3–LC3B and either GFP-tagged HsSTX17TM, DsSTX17TM, or CeSTX17TM were cultured in starvation medium for 1 hr. GFP–STX17TM intensities of mRuby3–LC3B-positive ring-like structures were quantified (n>30). In box plots, solid horizontal lines indicate medians, boxes indicate the interquartile ranges (25th to 75th percentiles), whiskers indicate the 5th to 95th percentiles, and dots represent outliers. ( C ) MEFs stably expressing one of the GFP-tagged alanine replacement mutants (shown in ) and mRuby3–LC3B were cultured in starvation medium for 1 hr. Quantification results are shown in . ( D ) MEFs stably expressing one of the GFP-tagged charge replacement mutants (shown in ) and mRuby3–LC3B were cultured in starvation medium for 1 hr. Quantification results are shown in . Experiments were performed three times independently. Scale bars, 10 μm (main), 1 μm (inset) ( B, C, and D ).

Article Snippet: First, cDNAs encoding human STX17 , D. melanogaster STX17 (NM_079202), C. elegans STX17 (NM_059941), rat LC3B , and mouse ATG5 (NM_053069) were inserted into pMRXIP (harboring a puromycin-resistant marker) ( ; ), pMRXIZ (harboring a zeocin-resistant marker) , and pMRXIB (harboring a blasticidin-resistant marker) ( ) together with enhanced GFP or mRuby3 (codon-optimized, Addgene #74252).

Techniques: Sequencing, Stable Transfection, Expressing, Cell Culture

Journal: eLife

Article Title: Syntaxin 17 recruitment to mature autophagosomes is temporally regulated by PI4P accumulation

doi: 10.7554/eLife.92189

Figure Lengend Snippet: ( A and B ) Mouse embryonic fibroblasts (MEFs) stably expressing the indicated GFP-tagged PI4P probe, CERT(PHD)(W33A), FAPP(PHD), OSBP(PHD) or P4M-SidMx2, and mRuby3–STX17TM or mRuby3–ATG5 were cultured in starvation medium for 1 hr. ( C ) GFP intensities of mRuby3-positive structures (n>50) in ( B ) were quantified. In box plots, solid horizontal lines indicate medians, boxes indicate the interquartile ranges (25th to 75th percentiles), whiskers indicate the 5th to 95th percentiles, and dots represent outliers. Differences were statistically analyzed by Welch’s t -test. ( D ) Time-lapse analysis of MEFs stably expressing GFP–CERT(PHD) cultured in starvation medium containing LysoTracker Deep Red. ( E ) U2OS cells stably expressing GFP–CERT(PHD) and mRuby3–LC3B were transfected with siSTX17 and siYKT6. After 3 days, cells were cultured in starvation medium for 1 hr, and immunostained with anti-LAMP1 antibodies. ( F ) WT and ATG8 hexa KO HeLa cells stably expressing GFP–STX17TM and transiently expressing mRuby3–CERT(PHD) were cultured in starvation medium for 1 hr. Experiments were performed three times independently. Scale bars, 10 μm ( A, B, and E [main]), 1 μm ( A, B, E [inset], and D ).

Article Snippet: First, cDNAs encoding human STX17 , D. melanogaster STX17 (NM_079202), C. elegans STX17 (NM_059941), rat LC3B , and mouse ATG5 (NM_053069) were inserted into pMRXIP (harboring a puromycin-resistant marker) ( ; ), pMRXIZ (harboring a zeocin-resistant marker) , and pMRXIB (harboring a blasticidin-resistant marker) ( ) together with enhanced GFP or mRuby3 (codon-optimized, Addgene #74252).

Techniques: Stable Transfection, Expressing, Cell Culture, Transfection

Journal: eLife

Article Title: Syntaxin 17 recruitment to mature autophagosomes is temporally regulated by PI4P accumulation

doi: 10.7554/eLife.92189

Figure Lengend Snippet: ( A ) GFP–STX17TM translated in vitro was incubated with rhodamine-labeled liposomes containing the indicated concentrations of phospholipids: 70% phosphatidylcholine (PC), 20% phosphatidylethanolamine (PE), and 10% of either PE, phosphatidylserine (PS), phosphatidylinositol 3-phosphate (PI3P), or phosphatidylinositol 4-phosphate (PI4P). GFP intensities of liposomes are quantified and shown as in (n>30). ( B ) GFP–STX17TM translated in vitro was incubated with rhodamine-labeled liposomes containing 70% PC, 20% PE and 10% PI4P in the presence of 1 M NaCl or 1.2 M sucrose. GFP intensities of liposomes were quantified and shown as in (n>30). ( C ) Amino acid sequences of GFP-tagged membrane surface charge probes. The positively charged residues are shown in orange. The farnesylation motif is indicated in green. ( D and E ) Mouse embryonic fibroblasts (MEFs) stably expressing one of the GFP-tagged charge probes and mRuby3-STX17TM ( C ) or mRuby3–LC3B ( D ) were cultured in starvation medium for 1 hr. GFP intensities of mRuby3–STX17TM-positive ( C ) or mRuby3–LC3B-positive ( D ) ring-like structures were quantified (n>70). ( F and G ) Time-lapse analysis of MEFs stably expressing the GFP-tagged 1K8Q ( E ) or 5K4Q ( F ) charge probes and mRuby3–STX17TM or mRuby3–LC3B cultured in starvation medium. Autophagosomes are indicated by arrows. ( H ) Summary of electrostatic dynamics of autophagosome formation. In box plots, solid horizontal lines indicate medians, boxes indicate the interquartile ranges (25th to 75th percentiles), whiskers indicate the 5th to 95th percentiles, and dots represent outliers. Differences were statistically analyzed by Welch’s t -test ( B ) or one-way ANOVA followed by Sidak’s multiple comparison test ( A, C, and D ). Experiments were performed three times independently. Scale bars, 1 μm ( A, B, F, and G ). Figure 2—source data 1. Data used for graphs presented in .

Article Snippet: First, cDNAs encoding human STX17 , D. melanogaster STX17 (NM_079202), C. elegans STX17 (NM_059941), rat LC3B , and mouse ATG5 (NM_053069) were inserted into pMRXIP (harboring a puromycin-resistant marker) ( ; ), pMRXIZ (harboring a zeocin-resistant marker) , and pMRXIB (harboring a blasticidin-resistant marker) ( ) together with enhanced GFP or mRuby3 (codon-optimized, Addgene #74252).

Techniques: In Vitro, Incubation, Labeling, Liposomes, Membrane, Stable Transfection, Expressing, Cell Culture, Comparison

Journal: eLife

Article Title: Syntaxin 17 recruitment to mature autophagosomes is temporally regulated by PI4P accumulation

doi: 10.7554/eLife.92189

Figure Lengend Snippet: ( A ) Mouse embryonic fibroblasts stably expressing one of the GFP-tagged charge probes and mRuby3–LC3B were cultured in starvation medium for 1 hr. ( B ) Time-lapse analysis of MEFs stably expressing GFP–3K6Q and mRuby3–STX17TM or mRuby3–LC3B cultured in starvation medium. Autophagosomes are indicated by arrows. ( C ) Time-lapse analysis of MEFs stably expressing one of the GFP-tagged charge probes and mRuby3–ATG5 cultured in starvation medium. Autophagosomes are indicated by arrows. Experiments were performed three times independently. Scale bars, 10 μm (A [main]), 1 μm ( A [inset], B and C ).

Article Snippet: First, cDNAs encoding human STX17 , D. melanogaster STX17 (NM_079202), C. elegans STX17 (NM_059941), rat LC3B , and mouse ATG5 (NM_053069) were inserted into pMRXIP (harboring a puromycin-resistant marker) ( ; ), pMRXIZ (harboring a zeocin-resistant marker) , and pMRXIB (harboring a blasticidin-resistant marker) ( ) together with enhanced GFP or mRuby3 (codon-optimized, Addgene #74252).

Techniques: Stable Transfection, Expressing, Cell Culture

Journal: eLife

Article Title: Syntaxin 17 recruitment to mature autophagosomes is temporally regulated by PI4P accumulation

doi: 10.7554/eLife.92189

Figure Lengend Snippet: ( A ) Mouse embryonic fibroblasts (MEFs) stably expressing the indicated GFP-tagged phospholipid probe and mRuby3–STX17TM were cultured in starvation medium for 1 hr. The following phospholipid probes were used: phosphatidic acid (PA), Spo20(PABD); PS, Evectin-2; diacylglycerol (DAG), PKD C1ab; PI3P, 2×FYVE; PI4P, CERT(PHD); PI5P, ING2(PlantHD); PI(3,4)P 2 , TAPP1(PHD); PI(4,5)P 2 , PLCd1(PHD); PI(3,5)P 2 , TRPML1(PHD); and PIP 3 , Btk(PHD). ( B ) MEFs stably expressing GFP–CERT(PHD) or TRPML1(PHD) were cultured in starvation medium containing LysoTracker Deep Red for 1 hr. ( C ) MEFs stably expressing GFP–PI4KB or GFP–PI4K2A and mRuby3–LC3B were cultured in starvation medium for 1 hr. Experiments were performed three times independently. Scale bars, 10 μm (main), 1 μm (inset).

Article Snippet: First, cDNAs encoding human STX17 , D. melanogaster STX17 (NM_079202), C. elegans STX17 (NM_059941), rat LC3B , and mouse ATG5 (NM_053069) were inserted into pMRXIP (harboring a puromycin-resistant marker) ( ; ), pMRXIZ (harboring a zeocin-resistant marker) , and pMRXIB (harboring a blasticidin-resistant marker) ( ) together with enhanced GFP or mRuby3 (codon-optimized, Addgene #74252).

Techniques: Stable Transfection, Expressing, Cell Culture

Journal: eLife

Article Title: Syntaxin 17 recruitment to mature autophagosomes is temporally regulated by PI4P accumulation

doi: 10.7554/eLife.92189

Figure Lengend Snippet: ( A ) Schematic representation of the in vitro autophagosome recruitment assay. Isolated autophagosomes were mixed with mGFP–STX17TM and either recombinant Sac1-phosphatase domain (Sac1PD) or its phosphatase-dead mutant (C392S). ( B ) Isolation of mature autophagosomes prior to their fusion with lysosomes. Homogenates of STX17 knockout HeLa cells stably expressing mRuby3–LC3B cultured in starvation medium at 1 hr were separated by the OptiPrep membrane flotation method. The autophagosome-containing fraction (#1: LC3-positive and LAMP1-negative) was collected. The positions of mRuby3–LC3B (black arrowhead) and endogenous LC3B (white arrowhead) are indicated. ( C ) Purification of recombinant yeast Sac1 (phosphatase domain, PD) and its phosphatase-dead (C392S) mutant and mGFP–STX17TM from High Five cells. ( D ) In vitro autophagosome association assay. Isolated autophagosomes were mixed with recombinant Sac1 (WT or C392S) for 30 min and then with mGFP–STX17TM for another 30 min. Representative images are shown. STX17-positivity rates were determined across three independent experiments (two of the three experiments were performed in a blind manner, and 80 autophagosomes were counted in each experiment). Solid horizontal lines indicate means. Differences were statistically analyzed by one-way ANOVA followed by Tukey’s test. The scale bar, 2.5 μm. Figure 4—source data 1. Data used for graphs presented in , . Figure 4—source data 2. Uncropped blot images of .

Article Snippet: First, cDNAs encoding human STX17 , D. melanogaster STX17 (NM_079202), C. elegans STX17 (NM_059941), rat LC3B , and mouse ATG5 (NM_053069) were inserted into pMRXIP (harboring a puromycin-resistant marker) ( ; ), pMRXIZ (harboring a zeocin-resistant marker) , and pMRXIB (harboring a blasticidin-resistant marker) ( ) together with enhanced GFP or mRuby3 (codon-optimized, Addgene #74252).

Techniques: In Vitro, Isolation, Recombinant, Mutagenesis, Knock-Out, Stable Transfection, Expressing, Cell Culture, Membrane, Purification, Histone Association Assay

Journal: eLife

Article Title: Syntaxin 17 recruitment to mature autophagosomes is temporally regulated by PI4P accumulation

doi: 10.7554/eLife.92189

Figure Lengend Snippet: ( A ) Mouse embryonic fibroblasts (MEFs) stably expressing GFP–CERT(PHD) and mRuby3–STX17TM or mRuby3–ATG5 were cultured in starvation medium for 1 hr. GFP intensities of mRuby3-positive structures (n>60) were quantified. In box plots, solid horizontal lines indicate medians, boxes indicate the interquartile ranges (25th to 75th percentiles), whiskers indicate the 5th to 95th percentiles, and dots represent outliers. Differences were statistically analyzed by Welch’s t -test. ( B–D ) Time-lapse analysis of MEFs stably expressing GFP–CERT(PHD) and mRuby3–ATG5 ( B ), WIPI2B–mRuby3 ( C ), or mRuby3–STX17TM and HaloTag–LC3B (visualized with SaraFluor 650T HaloTag ligand) ( D ) cultured in starvation medium. Autophagosomes are indicated by arrows. Experiments were performed three times independently. Scale bars, 10 μm ( A [main]), 1 μm ( A [inset], B–D ). Figure 3—source data 1. Data used for graphs presented in and .

Article Snippet: First, cDNAs encoding human STX17 , D. melanogaster STX17 (NM_079202), C. elegans STX17 (NM_059941), rat LC3B , and mouse ATG5 (NM_053069) were inserted into pMRXIP (harboring a puromycin-resistant marker) ( ; ), pMRXIZ (harboring a zeocin-resistant marker) , and pMRXIB (harboring a blasticidin-resistant marker) ( ) together with enhanced GFP or mRuby3 (codon-optimized, Addgene #74252).

Techniques: Stable Transfection, Expressing, Cell Culture

Journal: eLife

Article Title: Syntaxin 17 recruitment to mature autophagosomes is temporally regulated by PI4P accumulation

doi: 10.7554/eLife.92189

Figure Lengend Snippet: HEK293T cells stably expressing mRuby3–STX17TM ( A ) or mRuby3–CERT(PHD) ( B ) and HaloTag-LC3B were cultured in starvation medium for 1 hr and then treated with and without 10 μM NC03 for 10 min. Representative confocal images are shown. STX17TM- or CERT(PHD)-positive rates of LC3B structures per cell (n>20 cells) are shown in the graphs. Solid horizontal lines indicate medians, boxes indicate the interquartile ranges (25th to 75th percentiles), whiskers indicate the 5th to 95th percentiles, and dots represent outliers. Differences were statistically analyzed by Welch’s t -test. Scale bars, 10 μm (main), 1 μm (inset).

Article Snippet: First, cDNAs encoding human STX17 , D. melanogaster STX17 (NM_079202), C. elegans STX17 (NM_059941), rat LC3B , and mouse ATG5 (NM_053069) were inserted into pMRXIP (harboring a puromycin-resistant marker) ( ; ), pMRXIZ (harboring a zeocin-resistant marker) , and pMRXIB (harboring a blasticidin-resistant marker) ( ) together with enhanced GFP or mRuby3 (codon-optimized, Addgene #74252).

Techniques: Stable Transfection, Expressing, Cell Culture

Journal: eLife

Article Title: Syntaxin 17 recruitment to mature autophagosomes is temporally regulated by PI4P accumulation

doi: 10.7554/eLife.92189

Figure Lengend Snippet:

Article Snippet: First, cDNAs encoding human STX17 , D. melanogaster STX17 (NM_079202), C. elegans STX17 (NM_059941), rat LC3B , and mouse ATG5 (NM_053069) were inserted into pMRXIP (harboring a puromycin-resistant marker) ( ; ), pMRXIZ (harboring a zeocin-resistant marker) , and pMRXIB (harboring a blasticidin-resistant marker) ( ) together with enhanced GFP or mRuby3 (codon-optimized, Addgene #74252).

Techniques: Sequencing, Staining, Recombinant, Plasmid Preparation, Expressing, Software

Journal: Cell reports

Article Title: Combination PD-1 and PD-L1 Blockade Promotes Durable Neoantigen-Specific T Cell-Mediated Immunity in Pancreatic Ductal Adenocarcinoma

doi: 10.1016/j.celrep.2019.07.059

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Real time PCR was performed in triplicate on a BioRad CFX96 Touch Real-Time PCR Detection System by measuring SYBR Green (BioRad) fluorescence for 40 cycles.

Techniques: Blocking Assay, Recombinant, Luciferase, Derivative Assay, Red Blood Cell Lysis, Electron Microscopy, SYBR Green Assay, Clone Assay, Retroviral, Plasmid Preparation, Software, Cell Counting, Magnetic Beads