Journal: Viruses

Article Title: Porcine Circovirus Type 2 Activates CaMMKβ to Initiate Autophagy in PK-15 Cells by Increasing Cytosolic Calcium

doi: 10.3390/v8050135

Figure Lengend Snippet: Inhibition of CaMKI by KN93 or RNA interference reduced expression of LC3-II and Cap proteins without affecting WIPI1. PK-15 cells were infected with PCV2 (MOI ≈ 1) in the presence of KN93 (2 μM) or siRNA targeting CaMKI (siCaMKI). Cells were collected at 36 hpi and the lysates were subjected to Western blotting. ( A ) Representative images of Western blotting for target proteins from cells treated with KN93; ( B ) Ratios of WIPI1, p-CaMKI, Cap and LC3-II to β-actin; ( C ) Representative images of Western blotting for target proteins from cells treated with siCaMKI; ( D ) Ratios of WIPI1, t-CaMKI, p-CaMKI, Cap and LC3-II to β-actin. Ratios of targeted proteins to β-actin were normalized to mock infection set at 1.0. Data are reported as the mean ± SEM of three independent experiments (ns p > 0.05; * p < 0.05; and ** p < 0.01).

Article Snippet: The chemicals used included: mTOR inhibitor rapamycin (Rapa) (Merck, Darmstadt, Germany), CaMKKβ inhibitor 7-Oxo-7H-benzimidazo(2,1-a)benz(de)isoquinoline-3-carboxylic acid acetate acid (STO-609) (Sigma), CaMKI inhibitor 2-( N -(2-hydroxyethyl))- N -(4-methoxybenzenesulfonyl)amino- N -(4-chlorocinnamyl)- N -methylamine (KN93) (Selleck Chemicals LLC, Houston, TX, USA), Sarcoplasmic/endoplasmic reticulum Ca 2+ - adenosine triphosphatase (SERCA) inhibitor thapsigargin (TG) (Abcam, Cambridge, UK), phosphoinositide 3-kinase (PI3K) inhibitor wortmannin (WM) (Sigma), IP3R inhibitor 2-aminoethoxydiphenyl borate (2-APB) (Sigma), and IP3R activator d -myo-inositol 1,4,5-tris-phosphate trisodium salt (Sigma).

Techniques: Inhibition, Expressing, Infection, Western Blot

Journal: Viruses

Article Title: Porcine Circovirus Type 2 Activates CaMMKβ to Initiate Autophagy in PK-15 Cells by Increasing Cytosolic Calcium

doi: 10.3390/v8050135

Figure Lengend Snippet: Formation of WIPI1 puncta and colocalization of WIPI1 and LC3 were increased by PCV2 infection, but decreased by treatment with STO-609 or KN93. ( A ) PCV2-infected cells were transfected with pcDNA3.1-DsRed-WIPI1 at 6 hpi, treated with STO-609 (10 μM) or KN93 (2 μM) at 6 hours post-transfection (hpt), and incubated for additional 24 h. 0.5 μM thapsigargin (TG) or 1 μM wortmannin (WM) was used as positive or negative control, respectively. Cells were examined for formation of DsRed-WIPI1 puncta under the confocal microscope (scale bar, 10 μM) at 36 hpi; ( B ) average number of DsRed-WIPI1 puncta per cell from at least 50 cells/experiment in three independent experiments; ( C ) pcDNA3.1-DsRed-WIPI1 was transiently transfected into PCV2 pre-infected PK-15 cells stably expressing EGFP-LC3 (PK15/EGFP-LC3) at 6 hpi (MOI ≈ 1) and treated with STO-609 or KN93 as above. Representative confocal images of colocalization of DsRed-WIPI1 and EGFP-LC3 in PK15/EGFP-LC3 cells are shown (scale bar, 10 μM); and ( D ) average number of colocalization of DsRed-WIPI1 and EGFP-LC3 puncta per cell from at least 50 cells/experiment in three independent experiments. Data are reported as the mean ± SEM (ns, p > 0.05; * p < 0.05; and ** p < 0.01).

Article Snippet: The chemicals used included: mTOR inhibitor rapamycin (Rapa) (Merck, Darmstadt, Germany), CaMKKβ inhibitor 7-Oxo-7H-benzimidazo(2,1-a)benz(de)isoquinoline-3-carboxylic acid acetate acid (STO-609) (Sigma), CaMKI inhibitor 2-( N -(2-hydroxyethyl))- N -(4-methoxybenzenesulfonyl)amino- N -(4-chlorocinnamyl)- N -methylamine (KN93) (Selleck Chemicals LLC, Houston, TX, USA), Sarcoplasmic/endoplasmic reticulum Ca 2+ - adenosine triphosphatase (SERCA) inhibitor thapsigargin (TG) (Abcam, Cambridge, UK), phosphoinositide 3-kinase (PI3K) inhibitor wortmannin (WM) (Sigma), IP3R inhibitor 2-aminoethoxydiphenyl borate (2-APB) (Sigma), and IP3R activator d -myo-inositol 1,4,5-tris-phosphate trisodium salt (Sigma).

Techniques: Infection, Transfection, Incubation, Negative Control, Microscopy, Stable Transfection, Expressing

Journal: Developmental cell

Article Title: A mechanism coupling systemic energy sensing to adipokine secretion

doi: 10.1016/j.devcel.2017.09.007

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: See ELISA assay procedure below. table ft1 table-wrap mode="anchored" t5 REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies GFP-nAb ™ Agarose Allele Biotech ABP-nAb-GFPA050 Chicken anti-GFP Abcam ab13970 GFP coating antibody for ELISA Allele Biotech ACT-CM-GFPTRAP GFP detection antibody for ELISA Rockland 600-401-215 Rabbit anti-GRASP65 Abcam ab30315 Anti-phospho-Threnonine Cell Signaling 9381 Rabbit-anti-Lsp1-gamma ( Burmester et al., 1999 ) N/A Mouse monoclonal Anti-α-Tubulin Sigma T5168 Rabbit-anti-PLIN1 ( Beller et al., 2010 ) N/A Rabbit-anti-tRFP Evrogen AB233 Rabbit-anti-Dilp5 ( Geminard et al., 2009 ) N/A Bacterial and Virus Strains BL21(DE3)pLysS EMD Millipore 69451-3 NEB® 10-beta Competent E. coli NEB C3019H Biological Samples N/A Chemicals, Peptides, and Recombinant Proteins U73122 Tocris 1268 U73343 Tocris 4133 Brefeldin A1 Sigma B5936-200UL BAPTA-AM Sigma A1076-25MG Ionomycin Sigma I9657-1MG KN93 Santa Cruz Biotech sc-202199 KN92 Santa Cruz Biotech sc-311369 Effectene QIAGEN 301427 Gibco ™ Schneider’s Drosophila Sterile Medium ThermoFisher 21720024 SlowFade Gold antifade reagent with DAPI Invitrogen S36938 M3RM medium also known as Cl.8 ( Zartman et al., 2013 ) N/A Fetal Bovine Serum ThermoFisher 10437028 Gibco ™ Penicillin-Streptomycin (5,000 U/mL) ThermoFisher 15-070-063 Low melting agarose Invitrogen 16520-100 Activated recombinant CaMKII NEB P6060S Glycerol standard Sigma G7793-5ML Free glycerol Reagent Sigma F6428-40ML Triglyceride reagent Sigma T2449-10ML Recombinant GFP protein Vector Labs MB-0752 Ni-NTA His•Bind® Superflow ™ Resin EMD Millipore 70691-3 Recombinant 6XHis-MBP- Drosophila GRASP protein This paper N/A Gateway® LR Clonase® II Enzyme mix Invitrogen 11791-020 Gateway® BP Clonase II Enzyme mix Invitrogen 11789-020 MEGAscript® T7 Transcription Kit ThermoFisher AMB1334-5 Q5® Site-Directed Mutagenesis Kit NEB E0554S Critical Commercial Assays 1-step Ultra-TMB ELISA substrate Pierce 34028 Renilla-Glo® Luciferase reagent Promega E2710 Pro-Q® Diamond Phosphoprotein Blot Stain Thermo Fisher Scientific Inc P33356 Deposited Data N/A Experimental Models: Cell Lines D. melanogaster: Cell line S2R+ Laboratory of Norbert Perrimon N/A Experimental Models: Organisms/Strains UAS-GRASP::GFP BDSC 8507, 8508 UAS-myr::GFP BDSC 32197 UAS-Golgi-RFP BDSC 30908 Lpp-Gal4 ( Brankatschk and Eaton, 2010 ) N/A Df(3L)BSC552 BDSC 26502 Df(3L)BSC445 BDSC 24949 GRASP-RNAi TRiP.

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Plasmid Preparation, Mutagenesis, Luciferase, Staining, Amplification, Synthesized, Software, Mass Spectrometry