In order to detect [ROS]mito, the HT-22 cells were transfected with the plasmid pMitotimer (Addgene, Cambridge, MA, USA), an engineered reporter gene used to assess mitochondrial content, structure, stress, and damage39 (link), using Lipofectamine LTX-plus (Invitrogen, Carlsbad, CA, USA). Images were acquired at the following excitation/emission wavelengths (nm): pMitotimer green (488/518) and red (543/572), and analyzed using Image J (National Institutes of Health, Bethesda, MD, USA). The level of [ROS]mito was reported as the ratio of red/green as previously described39 (link).
Pmitotimer
Manufactured by Addgene
Sourced in United States
PMitoTimer is a fluorescent protein that enables visualization of mitochondrial protein folding and dynamics in living cells. It undergoes a time-dependent shift in fluorescence emission from green to red, allowing monitoring of mitochondrial protein maturation and turnover.
Automatically generated - may contain errors
Lab products found in correlation
Opti mem, by Thermo Fisher Scientific (2 mentions)
Lipofectamine ltx plus, by Thermo Fisher Scientific (1 mentions)
Renilla luciferase plasmid, by Promega (1 mentions)
Lysotracker, by Thermo Fisher Scientific (1 mentions)
Mitotracker, by Thermo Fisher Scientific (1 mentions)
Hoechst, by Thermo Fisher Scientific (1 mentions)
Mitotracker deep red, by Thermo Fisher Scientific (1 mentions)
Pegfp n1 vector, by Takara Bio (1 mentions)
Escherichia coli dh5a, by Thermo Fisher Scientific (1 mentions)
Lamp1 mneongreen, by Addgene (1 mentions)
Phyper dmito, by Evrogen (1 mentions)
Midiprep kit, by Qiagen (1 mentions)
Plentiekar2g2, by Addgene (1 mentions)
Gw1 phred, by Addgene (1 mentions)
Ptriex rhoa flare, by Addgene (1 mentions)
Egfp p65, by Addgene (1 mentions)
Mcherry lifeact 7, by Addgene (1 mentions)
Pgp cmv gcamp6f, by Addgene (1 mentions)
Prk5 myc rhoa t19n, by Addgene (1 mentions)
Megfp htrak1, by Addgene (1 mentions)
9 protocols using pmitotimer
1
Mitochondrial ROS Assessment in HT-22 Cells
2
Dengue Protein Plasmid Construction
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The dual fluorescent reporter plasmids pAT016 (p-mito-mRFP-EGFP, a kind gift from Andreas Till, and pTF-LC3, a kind gift from Tamotsu Yoshimori) were used to monitor mitophagy and autophagy flux, respectively (18 (link), 19 (link)). pMito-timer (Addgene no. 52659) was used to track mitochondrial biogenesis. PGC-1 alpha promoter 2-kb luciferase plasmid (Addgene no. 8887) and renilla luciferase plasmid (Promega no. E2231) were obtained from Addgene and Promega, respectively. The dengue 2 nonstructural protein constructs in the pLVX-IRES-ZsGreen1 backbone were a gift from Ali Amara and have been described previously (80 (link)). The plasmids were used to subclone the nonstructural proteins into the pCMV3Tag3A vector by restricted digestion with either SpeI-BamHI (NS1, NS2B, and NS3) or EcoRI-BamHI (NS2A, NS5, NS4A, or NS4B) and ligation into pCMV3Tag3A. The dengue 2 structural proteins were cloned into the pCMV3Tag3A vector using gene-specific primers with suitable restriction sites. The cloning primers for PCR synthesis of envelope, prM, and capsid regions used are shown in Table S1. The PCR fragments were restriction digested, gel purified, and ligated into the pCMV3Tag3A vector. The positive clones were confirmed by DNA sequencing.
3
Fluorescent Imaging of Mitochondrial Dynamics
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Fluorescent reporter assays were performed on live HC11 and CIT3 cells at the Image Analysis Laboratory at Texas A&M University on a Zeiss LSM 780 NLO Multiphoton microscope with a 63× plan apochromat objective. The pMitoTimer construct (Addgene, 52659, deposited by the Zhen Yan Laboratory) was transiently transfected in HC11 control and Sim2s cells, as previously described [38 (link)]. Fluorescent images were collected beginning at 24 h of priming and proceeding through 48 h of differentiation. For image analysis, we excluded saturated pixels (gray level = 225) and measured single channel mean fluorescent intensities (MFI). The MFI for RFP was divided by the MFI for GFP to generate a red:green ratio for each image. A minimum of 10 images were evaluated per sample. Average ratios were then compared between groups. MitoTracker, LysoTracker, and MitoTracker DeepRed (Life Technologies) staining was performed for 30 min at 37 °C in phenol red-free media. After dye incubation, cells were stained with 16 mM Hoechst (Thermo Fisher Scientific), washed with PBS, and a minimum of 10 images were collected for each sample. Image analysis was performed with ImageJ, and saturated pixels were excluded. MFI of each dye was calculated and compared between groups.
4
Constructing Mitochondrial Targeted Plasmids
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To construct a mitochondrially targeted GFP (mtGFP) expression plasmid, the egfp were amplified from the pEGFP-N1 vector (Takara Bio Inc.). This was cloned into a modified pcDNA3.1 vector downstream of the mitochondria targeting presequence to generate the necessary constructs. For the Drp1 overexpression vector, the Drp1 insert was amplified from HUVEC cDNA using the following primers: forward, 5′-TCAGGCGGCCGCAGCGCATGGCCTGCCGGGA-3′, and reverse, 5′-CTAGCTCGAGCTACTCTATACGGTTATGTTCCAAAG-3′. The product was ligated into an empty pcDNA3.1 vector.
To generate GFP-P53 fusion construct, P53 was amplified from the pCDNA3-HA-p53 (Marin et al., 2000 (link)) and cloned into the pCDNA3 vector already containing a GFP gene, so the product represented a p53 fused with its amino terminus to the GFP, including two linker amino acids between the proteins originating from the EcoRI restriction site. pcDNA3-Drp1K38A was a gift from A. van der Bliek (David Geffen School of Medicine at University of California, Los Angeles, South Los Angeles, CA) and R. Youle (National Institute of Neurological Disorders and Stroke, Porter Neuroscience Research Center, Bethesda, MD; plasmid 45161; Addgene). pMitoTimer was a gift from Z. Yan (University of Virginia, Charlottesville, VA; plasmid 52659; Addgene).
To generate GFP-P53 fusion construct, P53 was amplified from the pCDNA3-HA-p53 (Marin et al., 2000 (link)) and cloned into the pCDNA3 vector already containing a GFP gene, so the product represented a p53 fused with its amino terminus to the GFP, including two linker amino acids between the proteins originating from the EcoRI restriction site. pcDNA3-Drp1K38A was a gift from A. van der Bliek (David Geffen School of Medicine at University of California, Los Angeles, South Los Angeles, CA) and R. Youle (National Institute of Neurological Disorders and Stroke, Porter Neuroscience Research Center, Bethesda, MD; plasmid 45161; Addgene). pMitoTimer was a gift from Z. Yan (University of Virginia, Charlottesville, VA; plasmid 52659; Addgene).
5
Plasmid Constructs for Organelle Imaging
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All DNA constructs were produced using Escherichia coli DH5a (Thermo Fisher Scientific) and extracted using a plasmid midiprep kit from Qiagen. The plasmids used in this study were: RFP-Galectin-321 , pMitoTimer (Addgene, 52659), pHyPer-dMito (Evrogen, FP942), CystatinB C-GFPSpark (HG10435-ACG, Sino Biological), Lamp1-mNeonGreen (Addgene, 98882), RAB7-GFP54 (link) and RAB7(QG7L)-GFP54 (link). The mCherry-EGFP-NIPSNAP mitophagy reporter was kindly provided by Dr. Anne Simonsen (University of Oslo, Norway).
6
Comprehensive RhoA Signaling Toolkit
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Raichu-RhoA (provided by M. Matsuda [27 (link)]), pTriEx-RhoA FLARE.sc Biosensor WT (RRID:Addgene_12150), pTriEx-RhoA FLARE.sc Biosensor Q63L (RRID:Addgene_12151), pTriEx-RhoA FLARE.sc Biosensor T19N (RRID:Addgene_12152), pRK5-myc-RhoA WT (RRID:Addgene_12962), pRK5-myc-RhoA Q63L (RRID:Addgene_12964), pRK5-myc-RhoA T19N (RRID:Addgene_12963), EGFP-p65 (RRID:Addgene_111190), GW1-pHRed (RRID:Addgene_31473), GW1CMV-Perceval (RRID:Addgene_21737), Laconic/pcDNA3.1 (+) (RRID:Addgene_118627), Pyronic /pcDNA3.1 (+) (RRID:Addgene_51308), pcDNA3.1 FLII12Pglu-700uDelta6 (RRID:Addgene_17866), Cyto-ABKAR (RRID:Addgene_61510), pLentiEKAR2G2 (RRID:Addgene_40178), Kras-Src FRET biosensor (RRID:Addgene_78302), pFRET-HSP33 cys (RRID:Addgene_16076), pGP-CMV-GCaMP6F (RRID:Addgene_40755), mCherry-Lifeact-7 (RRID:Addgene_54491), pMitoTimer (RRID:Addgene_52659), pCLBW cox8 EGFP mCherry (RRID:Addgene_78520).
7
Molecular Tools for Organelle Dynamics
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Plasmids pLV-mitoDsRed (# 44386), pmCherry C1 Omp25 (#157758), and meGFP-hTRAK1 (# 188664) were purchased from Addgene. The coding regions of hOPTN (Addgene, #23053) and hOPTNΔC (Addgene, #23053) were cloned into meGFP-hTRAK1 to generate meGFP-hOPTN and meGFP-hOPTNΔC. The coding region of KIF5B 71 , TRAK1 (Addgene, #127621), his-hOPTN (Addgene, #23053), mOPTN (mouse tissue cDNA), pMitoTimer (Addgene, #52659), 4xmts-mScarlet-I (Addgene, #98818), TurboID (Addgene, #107169) and Cre 33 (link) were cloned and packaged into our pAM-AAV-mSncg-WPRE backbone containing the RGC-specific full-length mSncg promoter 33 (link). Cre was cloned into pAM-AAV-mSncg-EGFP-WPRE to generate AAV-mSncg-Cre-T2A-EGFP. The detailed procedure of the AAV production has been described previously 33 (link). Briefly, AAV plasmids containing the target genes were co-transfected with pAAV2 (pACG2)-RC triple mutant (Y444, 500, 730F) and the pHelper plasmid (StrateGene) into HEK293T cells by the PolyJet (SignaGen Laboratories, SL100688) transfection reagent. After transfection for 72 hours, the cells were lysed and purified by two rounds of cesium chloride density gradient centrifugation. The AAV titers of target genes were determined by real-time PCR and diluted to 1.5 x 1012 vector genome (vg)/ml for intravitreal injection.
8
Monitoring Mitophagy and Autophagy in A549 Cells
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pMitoTimer and pMRX-IP-GFP-LC3-RFP were obtained from AddGene. Briefly, A549 cells (3 × 105 cells/well) were seeded into 6-well plates (SPL Life Sciences) and incubated overnight. The following day, cells were treated with different concentrations of SRM 1648a urban particulate matter suspension (5, 10, 20, and 40 μg/cm2) and were transfected with either plasmid at 2 μg/well using polyethylenimine (PEI) and Opti-Mineral Essential Medium for 24 h incubation at 37°C and 5% CO2. All transfection experiments were performed in duplicate. The transfected cells were quantitated by fluorescence microscopy (Leica DMi8) for both green (excitation 420–460/emission 602–682 nm) and red (excitation 550–570/emission 602–682 nm) channels.
9
Mitochondrial Fluorescence Dynamics
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pMitoTimer (Addgene) incorporates a fluorescent timer reporter to mitochondria (pDsRed2-Mito) that fluoresces GFP when newly synthetized and irreversibly shifts to red spectrum (excitation/emission [Ex/Em] 558/583 nm) over time (28 (link)). MIO-M1 cells were incubated (12 hours) with a mixture of 50 ng plasmid DNA and 0.15 μL endofectin (GeneCopoeia) in Opti-MEM (Thermo Fisher Scientific). Cells were then maintained in NG, HG, or LG and fixed for microscopy.
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