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Pmrfp lc3

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PmRFP-LC3 is a plasmid that expresses the fusion protein of mRFP (monomeric red fluorescent protein) and the autophagy marker LC3 (microtubule-associated protein 1 light chain 3). This plasmid can be used to study the dynamics of autophagy in live cells.

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Lab products found in correlation

Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions) Mkeima red mito 7, by Addgene (2 mentions) Pact2, by Takara Bio (1 mentions) Pcr xl topo, by Thermo Fisher Scientific (1 mentions) Pdest27, by Thermo Fisher Scientific (1 mentions) Pegfp n1 vector, by Takara Bio (1 mentions) Pgbkt7, by Takara Bio (1 mentions) Pbabe puro mcherry egfp lc3b, by Addgene (1 mentions) Pn3 3xflag control, by Addgene (1 mentions) Pbabe egfp, by Addgene (1 mentions) Ecfp ssh1δc, by Addgene (1 mentions) Poptn egfp, by Addgene (1 mentions) Ha ubiquitin, by Addgene (1 mentions) Mrfp ubiquitin, by Addgene (1 mentions) Pcmv6 ac gfp, by OriGene (1 mentions) 4 kda fitc dextran, by Merck Group (1 mentions) Pmrfp egfp lc3, by Addgene (1 mentions) Zoe fluorescent cell imager, by Bio-Rad (1 mentions) Fluorescently conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions) Lysotracker red dnd 99, by Thermo Fisher Scientific (1 mentions)

14 protocols using pmrfp lc3

1

Cloning of EV-A71 Viral Proteins

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2B, 2C, 2BC, 3A, and 3AB genes of EV-A71 were chemically synthesized from GenScript and cloned into the BamHI and EcoRI sites of pEGFP-N1 vector (Clontech, Mountain View, CA, USA) to generate GFP fusion proteins. The STX17 gene was cloned into pGBKT7 (Clontech) and pCherry (Addgene, Cambridge, MA, USA).) vectors while the 2BC gene of EV-A71 was cloned into the pACT2 (Clontech) and pDEST27 (Thermo, Waltham, MA, USA) vectors. The pmRFP-LC3 and LAMP1-YFP vectors were obtained from Addgene. The tandem tagRFP-eGFP-LC3 vector was purchased from Thermo. The EV-A71 41-eGFP infectious cDNA clone was constructed by cloning the full-length genome of the virus with eGFP into pCR-XL-TOPO (Invitrogen, Carlsbad, CA, USA) as previously described [23 (link)].
Lai J.K., Sam I.C., Verlhac P., Baguet J., Eskelinen E.L., Faure M, & Chan Y.F. (2017). 2BC Non-Structural Protein of Enterovirus A71 Interacts with SNARE Proteins to Trigger Autolysosome Formation. Viruses, 9(7), 169.
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2

Plasmid-based Autophagy Monitoring

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The expression plasmids pmRFP-LC3 and pmRFP-GFP-LC3 were obtained from Addgene (Cambridge, MA). GFP-PRIP1 was prepared as previously described [16] (link).
Harada-Hada K., Harada K., Kato F., Hisatsune J., Tanida I., Ogawa M., Asano S., Sugai M., Hirata M, & Kanematsu T. (2014). Phospholipase C-Related Catalytically Inactive Protein Participates in the Autophagic Elimination of Staphylococcus aureus Infecting Mouse Embryonic Fibroblasts. PLoS ONE, 9(5), e98285.
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3

Autophagy and Actin Dynamics Regulation

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pN3-3xFlag-Control (Addgene, 107717, Guntram Suske Lab [75 (link)]); pmRFP-LC3 (Addgene, 21075, Tamotsu Yoshimori Lab [43 (link)]); pMXs-puro GFP-Sqstm1/p62 (Addgene, 38277, Noboru Mizushima Lab [49 (link)]); pBABE-puro mCherry-EGFP-LC3B (Addgene, 22418, Jayanta Debnath Lab [76 (link)]); pBABE-EGFP (Addgene, 36999, Debu Chakravarti Lab [77 (link)]); ECFP-SSH1ΔC (N461) (Dr. Mizuno lab [51 (link)]); EGFP-SSH1 (Dr. Storz lab [69 (link)]); mKeima-Red-Mito-7 (Addgene, 56018); pOPTN-EGFP (Addgene, 27052Beatrice Yue Lab [78 (link)]); pMXs-puro GFP-Sqstm1/p62ΔC (Addgene, 38282, Noboru Mizushima Lab [49 (link)]); pMXs-puro GFP-Sqstm1/p62 D337, 338, 339A (GFP-SQSTM1-LIR) (Addgene, 38280, Noboru Mizushima Lab [49 (link)]); HA-Ubiquitin (Addgene, 18712, Edward Yeh Lab [79 (link)]); pDR125 (Addgene, 37150, Dale Ramsden Lab); pN3-3xFlag-SSH1, mCherry-EGFP-Sqstm1/p62, mCherry-EGFP-Sqstm1S403A, mCherry-EGFP-Sqstm1S403E, pN3-Flag-SSH1ΔC, pN3-Flag-SSH1ΔN and pN3-Flag-SSH1ΔNC393S were generated in this work (see methods below).
Fang C., Woo J.A., Liu T., Zhao X., Cazzaro S., Yan Y., Matlack J., Kee T., LePochat P, & Kang D.E. (2020). SSH1 impedes SQSTM1/p62 flux and MAPT/Tau clearance independent of CFL (cofilin) activation. Autophagy, 17(9), 2144-2165.
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4

Generation of Protein Expression Constructs

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The pEGFP-N1 vectors containing human SOD1wt, SOD1A4V and G93A SOD1 were generated as described65 (link). Expression vector pCMV6-AC-GFP containing human TDP-43 and FUS was obtained from OriGene (USA). SOD1-tomato constructs were generated by replacing the GFP sequences in the GFP-tagged constructs65 (link) with the tomato red fluorescent protein. TDP-43wt-tomato (Addgene plasmid 28205, provided by Zuoshang Xu34 (link)), mRFP-Ubiquitin (Addgene plasmid 11935, provided by Nico Dantuma66 (link)) and pmRFP-LC3 (Addgene plasmid 21075, Provided by Tamotsu Yoshimori67 (link)) constructs were acquired from Addgene (USA). The vectors describing Httex1(46Q) fusions (C-terminally) to mCherry and Cerulean were generated as described33 (link)68 (link).
Farrawell N.E., Lambert-Smith I.A., Warraich S.T., Blair I.P., Saunders D.N., Hatters D.M, & Yerbury J.J. (2015). Distinct partitioning of ALS associated TDP-43, FUS and SOD1 mutants into cellular inclusions. Scientific Reports, 5, 13416.
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5

Autophagy Imaging in Sertoli Cells

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Sertoli cells (15 P-1) were supplemented with 1 mg/ml FITC-Dextran 4 kDa (Sigma) overnight (37o C, 5% CO2). Cells are transfected for 24 h using Attractene and 500 μg pmRFP-LC3 (AddGene, US20 (link)). Some of the cells were starved for 4 h in serum-free, low glucose DMEM (Sigma). The cells were challenged with LPS or iE-DAP and imaged live on ZOE Fluorescent Cell Imager (Bio-Rad, USA). Other cells were transfected for 24 h using Attractene and 500 μg pmRFP-EGFP-LC3 (ptfLC3) (AddGene, US20 (link)) and challenged with LPS or iE-DAP. Colocalization ratio was estimated using ImageJ Colocalization module (Pearson’s correlation coefficient) as Rtotal after automatically setting channel thresholds (using Costes method) and estimating the Manders coefficients.
Hayrabedyan S., Todorova K., Jabeen A., Metodieva G., Toshkov S., Metodiev M.V., Mincheff M, & Fernández N. (2016). Sertoli cells have a functional NALP3 inflammasome that can modulate autophagy and cytokine production. Scientific Reports, 6, 18896.
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6

Antibodies and Plasmid Constructs for Autophagy Research

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Antibodies were obtained from the following sources: rabbit anti-LC3 (Novus Biologicals, catalog #NB100-2331; Cell Signaling Technology, catalog #3868), mouse anti-β-actin (Sigma, catalog #S0644), mouse anti βIII-tubulin (Covance, catalog #MMS-435P), and rabbit anti-LAMP1 (Sigma, catalog #L1418; Abcam, catalog #ab24170). pEGFP-LC3 (plasmid 21073; Kabeya et al., 2000 (link)) and pmRFP-LC3 (plasmid 21075; Kimura et al., 2007 (link)) were generated in the laboratory of Tamotsu Yoshimori (Osaka University, Japan) and obtained from Addgene. The BoNT/A-Lc was subcloned into pEGFP-N1 to make pEGFP-BoNT/A-Lc from the pCMV-BoNT/A-Lc construct (a gift from Thomas Binz, Institut für Biochemie, Medizinische Hochschule Hannover, Hannover, Germany), FITC-conjugated p75NTR monoclonal antibody (Matusica et al., 2013 (link)) was a gift from Elizabeth Coulson (Queensland Brain Institute, University of Queensland, Brisbane, Australia). BoNT/A-truncated SNAP25 antibody was a kind gift from D. Sesardic (Division of Bacteriology, National Institute for Biological Standards and Control, Hertfordshire, United Kingdom). LysoTracker Red DND-99 was from Thermo Scientific (catalog #L-7528). Fluorescently conjugated secondary antibodies were obtained from Invitrogen. The remaining reagents were sourced from Sigma unless stated otherwise.
Wang T., Martin S., Papadopulos A., Harper C.B., Mavlyutov T.A., Niranjan D., Glass N.R., Cooper-White J.J., Sibarita J.B., Choquet D., Davletov B, & Meunier F.A. (2015). Control of Autophagosome Axonal Retrograde Flux by Presynaptic Activity Unveiled Using Botulinum Neurotoxin Type A. The Journal of Neuroscience, 35(15), 6179-6194.
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7

Optimizing Molecular Imaging Techniques

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ON-TARGETplus siRNAs for scrambled control (D-001810–10-20), mouse Bcl-2 (L-063933–00-0005), and mouse Fam134b (L-063361–01-0005) were purchased from Dharmacon. Cells were seeded in a 12-well plate for 24 h before transfection. Cells were transfected with siRNA (100 nM) using Lipofectamine 2000 (Invitrogen, 11,668–019) according to the manufacturer’s instructions. After 4 h, the medium was changed and the cells were incubated with fresh medium for 48 h.
For plasmid transfection, cells were transfected with 1 μg of plasmid using TurboFect Transfection Reagent (Thermo Fisher Scientific, R0534) according to the manufacturer’s instructions. The plasmids were as follows: pmRFP-GFP encoding tandem-fluorescent LC3 (mRFP-GFP-LC3; a gift from Dr. Inhee Mook-Jung, Seoul National University); pmRFP-LC3 (Addgene, #21075); pDsRed2-Mito (Clontech, #632421); pEGFP-DFCP1 (Addgene, #38269); pEGFP-Parkin was generated by subcloning Myc-Parkin from pRK5-Myc-Parkin (Addgene, #17612; Ted Dawson’s lab) into pEGFP-C1 (Clontech, #6084-1).
Lim Y., Kim S, & Kim E.K. (2021). Palmitate reduces starvation-induced ER stress by inhibiting ER-phagy in hypothalamic cells. Molecular Brain, 14, 65.
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8

Plasmid Constructs for Autophagy Studies

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Plasmids used in the study were as follows: ptfLC3 (mRFP-GFP-LC3; Addgene plasmid #21074) and pmRFP-LC3 (Addgene plasmid #21075) were gifts from Tamotsu Yoshimori (Osaka University). FLAG-Stx17 (Addgene plasmid #45911) and FLAG-SNAP29 (Addgene plasmid#45915) were gifts from Noburu Mizushima (The University of Tokyo). GFP-VAMP8 was a gift from Thierry Galli (Institute of Psychiatry and Neuroscience of Paris [IPNP]) (Addgene plasmid #42311; Paumet et al., 2000 (link)), mCherry-DFCP1 was a gift from Do-Hyung Kim (University of Minnesota) (Addgene plasmid #86746; Kim et al., 2015 (link)), and HA-hATG14 was a gift from Noburu Mizushima (Addgene plasmid #24294; Itakura et al., 2008 (link)). Plasmid-containing HA-VPS33A was a kind gift from Mahak Sharma, IISER Mohali. Myc-Stx17 plasmid was a kind gift from Viktor Korolchuk, Newcastle University. GFP-LC3 plasmid was generated in the lab by excising out mRFP fragment from mRFP-GFP-LC3 plasmid.
Vats S, & Manjithaya R. (2019). A reversible autophagy inhibitor blocks autophagosome–lysosome fusion by preventing Stx17 loading onto autophagosomes. Molecular Biology of the Cell, 30(17), 2283-2295.
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9

Plasmid Construction and Modification

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The plasmid EGFP-LC3 was kindly supplied by Jonathan C. Howard, Cologne, Germany and the LAMP1-GFP construct was a kind gift from John Brumell (Hospital for Sick Children). pmRFP-LC3 (plasmid 21075, provided by Tamotsu Yoshimori)52 (link) and GFP-Ubiquitin (plasmid 11928 provided by Nico Dantuma)53 (link) were obtained from Addgene. The plasmid CTSD-mRFP was kindly provided by François Darchen (CNRS, Université Paris Descartes)22 (link) All LGALS/galectin constructs were kindly provided by Felix Randow (MRC Laboratory of Molecular Biology). To generate the plasmid EGFP-LC3G120A, the G120A mutation was introduced into the LC3 gene in the plasmid EGFP-LC3 by using the 5′-GGGCTCGAGATGCCG-TCCGAGAAGACC-3′ and 5′-GGGGTCGACTTACACAGCCATTGCTGTCGCGAATGTCTC-3′ primers and the restriction enzymes XhoI (New England Biolabs, R0146S) and SacI (New England Biolabs, R0156S).
Prado M., Eickel N., De Niz M., Heitmann A., Agop-Nersesian C., Wacker R., Schmuckli-Maurer J., Caldelari R., Janse C.J., Khan S.M., May J., Meyer C.G, & Heussler V.T. (2015). Long-term live imaging reveals cytosolic immune responses of host hepatocytes against Plasmodium infection and parasite escape mechanisms. Autophagy, 11(9), 1561-1579.
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10

Neuronal Transfection for Autophagy and Mitochondrial Dynamics

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Neurons were cultured on 18 mm diameter coverslips pre-coated with poly-L-Lysine (Sigma-Aldrich, P-1524), and at 4 DIV, they were transfected with 2 µg of plasmid DNA per coverslip using Lipofectamine 2000 (Thermo Fisher Scientific, 11668-019) or Lipofectamine LTX (Thermo Fisher Scientific, 11668-019). A 1:3 ratio of µg plasmid DNA and µL lipofectamine was used. Neurons were incubated with the transfection mixture for 24 h. Plasmid pmRFP-LC3 (Addgene, #21075, Watertown, MA, USA) kindly provided by Dr. Susana Castro Obregón and mKeima-red-mito-7 (Addgene, #56018) were transfected.
Gómora-García J.C., Montiel T., Hüttenrauch M., Salcido-Gómez A., García-Velázquez L., Ramiro-Cortés Y., Gomora J.C., Castro-Obregón S, & Massieu L. (2023). Effect of the Ketone Body, D-β-Hydroxybutyrate, on Sirtuin2-Mediated Regulation of Mitochondrial Quality Control and the Autophagy–Lysosomal Pathway. Cells, 12(3), 486.
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