Journal: Developmental cell

Article Title: Single-Cell and Population-Level Analyses Using Real-Time Kinetic Labeling Couples Proliferation and Cell Death Mechanisms

doi: 10.1016/j.devcel.2019.08.016

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals, Peptides, and Recombinant Proteins ABT-737 Selleck Chemicals Cat#: S1002 mTNFα Peprotech Cat#: 315-01A mTRAIL Peprotech Cat#: 315-19 STYO21 Thermo Fisher Scientific Cat#: S7556 YOYO-3 Iodide Thermo Fisher Scientific Cat#: Y3606 zVAD-fmk APExBIO Cat#: A1902 Experimental Models: Cell Lines Bax +/+ Bak +/+ SV40 transformed MEF ATCC Cat#: CRL-2907; RRID: CVCL_U630 Bax −/− Bak −/− SV40 transformed MEF ATCC Cat#: CRL-2913; RRID: CVCL_U626 Cyld −/− SV40 transformed MEF Adrian Ting (Icahn School of Medicine at Mount Sinai, NY, USA) O’Donnell et al., 2011 Mlkl −/− adult ear fibroblasts Warren Alexander (University of Melbourne, Melbourne, Australia) Murphy et al., 2013 Recombinant DNA pProEx.Htb.annexinV Seamus Martin (Trinity College, Dublin, Ireland) Accession#: {"type":"entrez-nucleotide","attrs":{"text":"NM_001154","term_id":"1519243621","term_text":"NM_001154"}} NM_001154 ; Logue et al., 2009 Software and Algorithms Excel v16.16.9 Microsoft N/A ImageJ/Fiji v2.0.0-rc-69/1.52n National Institutes of Health N/A IncuCyte ZOOM v2018A Essen Biosciences N/A Prism v8.1.1 Graphpad Software N/A Sequential labeling pipeline This paper https://doi.org/10.5281/zenodo.3458574 Open in a separate window KEY RESOURCES TABLE SPARKL workflows use live-cell imagers to capture the kinetics of cell death in real time Multi-parametric analyses of cell death kinetics reveal mechanisms for comparative study Multiplex workflows accounts for cell-inherent or drug-induced proliferation changes Single-cell analyses quantify differential response to drugs in isogenic cell populations

Techniques: Recombinant, Transformation Assay, Software, Labeling

Journal: Neuron

Article Title: A Selectivity Filter Gate Controls Voltage-Gated Calcium Channel Calcium-Dependent Inactivation

doi: 10.1016/j.neuron.2019.01.011

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Bacterial and Virus Strains Dh5α competent E. coli MCLAB Cat#DA-196 Chemicals, Peptides, and Recombinant Proteins ethylene glycol-bis(β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA) Sigma-Aldrich Cat#E4378 1,2-Bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid tetrakis (BAPTA) Santa Cruz Biotechnology Cat#sc-202076 Dulbecco’s modified Eagle’s medium (DMEM) GIBCO Cat#11965-092 fetal bovine serum (FBS) GIBCO Cat#16140-071 L-glutamine GIBCO Cat#25030-081 penicillin/streptomycin UCSF Cell Culture Facility Cat#CCFGK004-153K01 Lipofectamine 2000 Invitrogen Cat#11668-019 Matrigel BD Biosciences Cat#354234 Critical Commercial Assays T7 mMessenger kit Thermo Fisher Scientific Cat# AM1344 QuikChange Site-Directed Mutagenesis Kit Agilent Cat#200515 Experimental Models: Cell Lines HEK293 ATCC Cat#CRL-1573 Xenopus oocytes This study N/A Experimental Models: Organisms/Strains Xenopus Laevis Nasco Cat# LM00531 Recombinant DNA human Ca V 1.2/pcDNA3.1 Findeisen and Minor, 2009 N/A human Ca V 1.2 D707A/pcDNA3.1 Shaya et al., 2014 N/A human Ca V 1.2 D707N/pcDNA3.1 Shaya et al., 2014 N/A human Ca V 1.2 D707G/pcDNA3.1 Shaya et al., 2014 N/A human Ca V 1.2 D707E/pcDNA3.1 This study N/A human Ca V 1.2 E1115A/pcDNA3.1 This study N/A human Ca V 1.2 D367A/pcDNA3.1 This study N/A human Ca V 1.2 E1119A/pcDNA3.1 This study N/A human Ca V 1.2 D1420A/pcDNA3.1 This study N/A human Ca V 1.2 G364D/D707G/pcDNA3.1 This study N/A human Ca V 1.2 D707G/G1116D/pcDNA3.1 This study N/A human Ca V 1.2 D707A/A1417D/pcDNA3.1 This study N/A rat Ca V 1.3 42a /pcDNA6/V5-His ABC Xu and Lipscombe, 2001 Addgene#26577 rat Ca V 1.3 D726A/pcDNA6/V5-His ABC This study N/A rat Ca V 1.3 E1121A/pcDNA6/V5-His ABC This study N/A human Ca V 2.1/pcDNA3.1 Kim et al., 2008 N/A human Ca V 2.1 D667A/pcDNA3.1 This study N/A human Ca V 2.1 E1461A/pcDNA3.1 This study N/A rat Ca V β 2a /pTracer-CMV2-GFP Findeisen and Minor, 2009 N/A rabbit Ca V β 3 /pSport This study N/A rabbit Ca V α 2 δ-1/pcDNA3.1 Findeisen and Minor, 2009 N/A sv40 T-antigen/Bluescribe addgene Cat#21826 Software and Algorithms pCLAMP 9 Molecular Devices N/A Clampfit 10 Molecular Devices N/A SigmaStat 3.1 Systat N/A Open in a separate window KEY RESOURCES TABLE Ca V selectivity filter forms the calcium-dependent inactivation (CDI) endpoint Conserved Ca V domain II selectivity filter (+1) aspartate plays an active role in CDI Ca V selectivity filter asymmetry is important for CDI Ca V s gating relies on an SF-based gating framework shared among the VGIC superfamily

Techniques: Recombinant, Modification, Cell Culture, Mutagenesis, Software

Journal: Journal of Innovative Optical Health Sciences

Article Title: Real-time detection of LMP1/LMP1 interaction in MβCD-induced apoptosis of nasopharyngeal carcinoma cells using FRET method

doi: 10.1142/s1793545819500196

Figure Lengend Snippet: Fig. 1. Cytotoxicity of MCD in CNE1-LMP1 cells and CNE1 cells. (a) Viability of CNE1-LMP1 cells, (b) viability of CNE1 cells, and (c) di®erences of viability between CNE1-LMP1 cells and CNE1 cells were measured by MTT assays after 40 mM MCD treatment for 30 min at 37C. Data represents the mean SD of at least three experiments. Di®erences between two groups were compared by Student's t-test.

Article Snippet: Plasmids and transfection LMP1 vector (p1990 SV40-LMP1) was a gift from Bill Sugden (Addgene plasmid #26654), cyan °uorescent protein (CFP) vector (EX-NEG-M33) and 1950019-2 J. I nn ov .

Techniques:

Journal: Journal of Innovative Optical Health Sciences

Article Title: Real-time detection of LMP1/LMP1 interaction in MβCD-induced apoptosis of nasopharyngeal carcinoma cells using FRET method

doi: 10.1142/s1793545819500196

Figure Lengend Snippet: Fig. 2. Induction of apoptosis in NPC cells by MCD treatment. (a) Expression of LMP1 protein in CNE1 and CNE1-LMP1 cells were measured by western blotting. (b) CNE1 cells were treated with 40 mM MCD for 30 min. (c) CNE1-LMP1 cells were treated with 40 mM MCD for 30 min. (d) Di®erences of caspase-3 cleavage ratio between CNE1 and CNE1-LMP1 cells. (e) Di®erences of mean °uorescence intensity induced by caspase-3 cleavage between CNE1 and CNE1-LMP1 cells. Corresponding statistical results from at least 4000 cells. These images were representative of three independent experiments. Scale bar, 100 m.

Article Snippet: Plasmids and transfection LMP1 vector (p1990 SV40-LMP1) was a gift from Bill Sugden (Addgene plasmid #26654), cyan °uorescent protein (CFP) vector (EX-NEG-M33) and 1950019-2 J. I nn ov .

Techniques: Expressing, Western Blot

Journal: Journal of Innovative Optical Health Sciences

Article Title: Real-time detection of LMP1/LMP1 interaction in MβCD-induced apoptosis of nasopharyngeal carcinoma cells using FRET method

doi: 10.1142/s1793545819500196

Figure Lengend Snippet: Fig. 4. Fluorescence spectra of CNE1 cells expressing fusion proteins. (a) CNE1 cells co-transfected with CFP and YFP plasmids, (b) transfected with CFP-YFP plasmids (18AA) and (c) co-transfected with LMP1-CFP and LMP1-YFP plasmids. All spectra were normalized as percentage of CFP maximal emission peak. These spectra were representative of three independent experiments.

Article Snippet: Plasmids and transfection LMP1 vector (p1990 SV40-LMP1) was a gift from Bill Sugden (Addgene plasmid #26654), cyan °uorescent protein (CFP) vector (EX-NEG-M33) and 1950019-2 J. I nn ov .

Techniques: Fluorescence, Expressing, Transfection

Journal: Journal of Innovative Optical Health Sciences

Article Title: Real-time detection of LMP1/LMP1 interaction in MβCD-induced apoptosis of nasopharyngeal carcinoma cells using FRET method

doi: 10.1142/s1793545819500196

Figure Lengend Snippet: Fig. 3. NF-kB p65 nuclear translocation in CNE1 and CNE1- LMP1 cells treated with 40 mM MCD for 15 min. These images were representative of three independent experiments. Scale bar, 20 m.

Article Snippet: Plasmids and transfection LMP1 vector (p1990 SV40-LMP1) was a gift from Bill Sugden (Addgene plasmid #26654), cyan °uorescent protein (CFP) vector (EX-NEG-M33) and 1950019-2 J. I nn ov .

Techniques: Translocation Assay

Journal: Journal of Innovative Optical Health Sciences

Article Title: Real-time detection of LMP1/LMP1 interaction in MβCD-induced apoptosis of nasopharyngeal carcinoma cells using FRET method

doi: 10.1142/s1793545819500196

Figure Lengend Snippet: Fig. 5. Analysis of LMP1/LMP1 interaction by FRET method. FRET was measured in CNE1 cells co-expressing the LMP1-CFP and LMP1-YFP fusion proteins as described in materials and methods. (a) FRET images. The pseudo-color images depicted FRET e±ciency, calculated as FRET/CFP ratio, with the color indicating the relative value at each pixel. (b) FRET °uorescence spectra. Images and °uorescence spectra were representative of three independent experiments. Scale bar, 10 m.

Article Snippet: Plasmids and transfection LMP1 vector (p1990 SV40-LMP1) was a gift from Bill Sugden (Addgene plasmid #26654), cyan °uorescent protein (CFP) vector (EX-NEG-M33) and 1950019-2 J. I nn ov .

Techniques: Expressing

Journal: Journal of Innovative Optical Health Sciences

Article Title: Real-time detection of LMP1/LMP1 interaction in MβCD-induced apoptosis of nasopharyngeal carcinoma cells using FRET method

doi: 10.1142/s1793545819500196

Figure Lengend Snippet: Fig. 6. Schematic ¯gure showing that relationship among lipid rafts, LMP1/LMP1 interaction and cell apoptosis.

Article Snippet: Plasmids and transfection LMP1 vector (p1990 SV40-LMP1) was a gift from Bill Sugden (Addgene plasmid #26654), cyan °uorescent protein (CFP) vector (EX-NEG-M33) and 1950019-2 J. I nn ov .

Techniques:

Journal: Neuron

Article Title: Thrombospondin-1 Mediates Axon Regeneration in Retinal Ganglion Cells

doi: 10.1016/j.neuron.2019.05.044

Figure Lengend Snippet: (A and B) Images of optic nerve sections showing CTB-labeled axons (grey) in C57BL/6J mice injected with either (A) AAV-THBS1 or (B) AAV-GFP. Asterisks, lesion site. Scale bars, 100 μm.

Article Snippet: To generate the pAAV.GFAP.SV40.Thbs1-HA.SV40(polyA) plasmid, the 377 bp CMV promoter coding sequence was deleted from pAAV.CMV.SV40.THBS1-HA.SV40(polyA) plasmid by digestion with AvrII and BspEI restriction enzymes. pAAV.GFAP.EGFP plasmid (Addgene #50473) was used as template to PCR amplify short GFAP promoter sequence using the following oligonucleotides: GFAP-F and GFAP-R.

Techniques: Labeling, Injection

Journal: Neuron

Article Title: Thrombospondin-1 Mediates Axon Regeneration in Retinal Ganglion Cells

doi: 10.1016/j.neuron.2019.05.044

Figure Lengend Snippet: (A) A schematic of THBS1 mutants investigated. All constructs contain the N-terminal signal peptide and have a C-terminal HA tag. Laminin G domain (LamG), oligomerization coiled coil (CC) domain, von Willebrand complex like domain (vWC), thrombospondin type 1 repeat domain (TSR1), epidermal growth factor-like repeat domains (EGF), type 3 repeat domain (TSR3), and the thrombospondin C-terminal domain (CTD). THBS4 is shown for comparison to THBS1.

Article Snippet: To generate the pAAV.GFAP.SV40.Thbs1-HA.SV40(polyA) plasmid, the 377 bp CMV promoter coding sequence was deleted from pAAV.CMV.SV40.THBS1-HA.SV40(polyA) plasmid by digestion with AvrII and BspEI restriction enzymes. pAAV.GFAP.EGFP plasmid (Addgene #50473) was used as template to PCR amplify short GFAP promoter sequence using the following oligonucleotides: GFAP-F and GFAP-R.

Techniques: Construct

Journal: Neuron

Article Title: Thrombospondin-1 Mediates Axon Regeneration in Retinal Ganglion Cells

doi: 10.1016/j.neuron.2019.05.044

Figure Lengend Snippet: (A) Images of optic nerve section showing GFP-labeled axons (green) from HB9:GFP;Bax−/− mice and CTB (magenta) following injection with AAV-THBS1 and optic nerve crush. Asterisks, lesion site.

Article Snippet: To generate the pAAV.GFAP.SV40.Thbs1-HA.SV40(polyA) plasmid, the 377 bp CMV promoter coding sequence was deleted from pAAV.CMV.SV40.THBS1-HA.SV40(polyA) plasmid by digestion with AvrII and BspEI restriction enzymes. pAAV.GFAP.EGFP plasmid (Addgene #50473) was used as template to PCR amplify short GFAP promoter sequence using the following oligonucleotides: GFAP-F and GFAP-R.

Techniques: Labeling, Injection

Journal: Neuron

Article Title: Thrombospondin-1 Mediates Axon Regeneration in Retinal Ganglion Cells

doi: 10.1016/j.neuron.2019.05.044

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: To generate the pAAV.GFAP.SV40.Thbs1-HA.SV40(polyA) plasmid, the 377 bp CMV promoter coding sequence was deleted from pAAV.CMV.SV40.THBS1-HA.SV40(polyA) plasmid by digestion with AvrII and BspEI restriction enzymes. pAAV.GFAP.EGFP plasmid (Addgene #50473) was used as template to PCR amplify short GFAP promoter sequence using the following oligonucleotides: GFAP-F and GFAP-R.

Techniques: shRNA, Recombinant, Multiplex Assay, Clone Assay, Software