Swiss 3T3 fibroblasts (Riken Cell Bank, Tsukuba, Japan) were cultured in 5% CO2 at 37°C in Dulbecco's modified Eagle's medium (Low-glucose DMEM; GIBCO, USA) supplemented with 10% fetal bovine serum (Nacalai Tesque, Japan) and penicillin/streptomycin (50 units/mL and 50 μg/mL, resp.) (GIBCO). For long-term observations of cell migration, the cells were plated on 35 mm glass-bottom dishes (Matsunami, Japan). For observation of actin SFs and FAs during cell migration, the cells were cotransfected with pAcGFP1-actin (Clontech, USA) and pTagRFP-zyxin (Evrogen, Russia) using FuGENE HD transfection reagents (Promega, USA) according to the manufacturer's instructions. The cells were then seeded on silicone gel substrates. Both glass-bottom dishes and silicone gel substrates were precoated with 50 μg/mL fibronectin (BD Biosciences, USA) for 30 minutes at room temperature. After plating, the cells were incubated for at least 3 h in a 5% CO2 incubator at 37°C, allowing the cells to adhere to and spread over the substrate.
Pacgfp1 actin
Manufactured by Takara Bio
Sourced in United States
PAcGFP1-Actin is a fluorescent protein-tagged actin construct that can be used to visualize actin dynamics in live cells. It is derived from the green fluorescent protein (GFP) and the Acanthastrea coral actin protein. PAcGFP1-Actin allows for the real-time monitoring of actin cytoskeleton reorganization and movement within the cellular environment.
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Lab products found in correlation
β actin, by Takara Bio (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Nacalai Tesque (1 mentions)
35 mm glass bottom dishes, by Matsunami (1 mentions)
Fugene hd transfection reagent, by Promega (1 mentions)
Low glucose dmem, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Fibronectin, by BD (1 mentions)
Pad cmv v5 dest, by Thermo Fisher Scientific (1 mentions)
Luciferin luciferase chemiluminescent method, by Roche (1 mentions)
Oligonucleotides, by Ajinomoto Group (1 mentions)
Pdsred monomer c1, by Takara Bio (1 mentions)
Pacgfp1 tubulin, by Takara Bio (1 mentions)
Wst 1 cell proliferation assay system, by Takara Bio (1 mentions)
Glass bottom dishes, by AGC Techno Glass (1 mentions)
6 protocols using pacgfp1 actin
1
Fibroblast Migration on Fibronectin-Coated Substrates
2
Fluorescent Protein Fusion Constructs
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Constructs used were: actin-eGFP (pAcGFP1-Actin, Clontech) and pEGFP (PT3051-5, Clontech). The pEGFP-C1-MK2-WT, pEGFP-C1-MK3-WT, pEGFP-C1-MK2-EE and pEGFP-C1-MK2-K79R constructs were generated as described previously17 (link)39 (link)40 (link).
3
Stable WI-38 Cell Line Creation
4
Constructing mCherry-tagged Rab and Actin Vectors
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All vectors are based on human adenovirus type 5 (Ad5) containing deletions in the E1 and E3 regions. The vector expressing mCherry-tagged α-tubulin, pAdEasy-mCherry-Tubulin [50] (link), was obtained from Addgene (#26767).
All other vectors were constructed by PCR amplification followed by Gateway recombination into pAd/CMV/V5-DEST (Invitrogen). We constructed an mCherry-tagged β-actin construct based on plasmid pAcGFP1-actin (Clontech). The coding sequences of Rab3a and Rab27a were kind gifts from M. Fukuda [51] (link). Rab5a and Rab11a were obtained from A. Ono, University of Michigan. Rab7a was obtained from R. Pagano [52] (link) via Addgene (#12605). Rab8a was obtained from M. Nachury [53] (link), via Addgene (#24898). In each case, mCherry was fused to the Rab GTPase as follows (the C-terminus of mCherry and the first methionine of the Rab are represented in bold type, and a linker peptide is in plain type): …MDELYK GTTLYTKVGSM MDELYK SGGSGGTGGSM
All other vectors were constructed by PCR amplification followed by Gateway recombination into pAd/CMV/V5-DEST (Invitrogen). We constructed an mCherry-tagged β-actin construct based on plasmid pAcGFP1-actin (Clontech). The coding sequences of Rab3a and Rab27a were kind gifts from M. Fukuda [51] (link). Rab5a and Rab11a were obtained from A. Ono, University of Michigan. Rab7a was obtained from R. Pagano [52] (link) via Addgene (#12605). Rab8a was obtained from M. Nachury [53] (link), via Addgene (#24898). In each case, mCherry was fused to the Rab GTPase as follows (the C-terminus of mCherry and the first methionine of the Rab are represented in bold type, and a linker peptide is in plain type): …
5
Measuring ATP Synthesis in 661W Cells
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ATP formation from ADP and inorganic phosphate (Pi) in 661W cells was measured by the luciferin/luciferase chemiluminescent method (Roche Applied Science), as described previously [38] . Cells (5 µg protein) were permeabilized with 0.03 mg/mL digitonin for 1 min, centrifuged for 9 min at 1000 rpm and resuspended in 50 mM Tris HCl (pH 7.4), 5 mM KCl, 1 mM EGTA, 5 mM MgCl 2 , 0.6 mM ouabain, 5 mM KH 2 PO 4 , 5 mM pyruvate and 2.5 mM malate and ampicillin (25 µg/mL). ATP synthesis was induced by adding 0.3 mM ADP.
2.9 β-actin and Mitochondrial Calcium Transfections 0.2 µg of each pAcGFP1-Actin (Clontech Laboratories, USA) and mito-GcaMP2 (Dr. Wang, Peking University) plasmids were used to transfect the cells for β-actin and mitochondrial calcium ([Ca 2+ ] mito ). Cells were transfected using Lipofectamine 2000 (Invitrogen, Germany) according to manufacturer's protocol. 4 h after transfection, complete growth medium was added and left undisturbed until the next morning.
2.9 β-actin and Mitochondrial Calcium Transfections 0.2 µg of each pAcGFP1-Actin (Clontech Laboratories, USA) and mito-GcaMP2 (Dr. Wang, Peking University) plasmids were used to transfect the cells for β-actin and mitochondrial calcium ([Ca 2+ ] mito ). Cells were transfected using Lipofectamine 2000 (Invitrogen, Germany) according to manufacturer's protocol. 4 h after transfection, complete growth medium was added and left undisturbed until the next morning.
6
Mammalian Cell Expression Vectors and Reagents
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The expression vectors in mammalian cells, pAcGFP1-Actin, pAcGFP1-Tubulin, and pDsRed-Monomer-C1, were obtained from Clontech. Oligonucleotides used as PCR primers in the construction of expression plasmids for fusion proteins were custom-synthesized by Gene Design (Osaka, Japan). The WST-1 cell proliferation assay system was purchased from Takara Bio (Kyoto, Japan). Lipofectamine 2000 was obtained from Invitrogen (Carlsbad, CA). Glass-bottom dishes used for cell culture and imaging experiments were purchased from AGC Techno Glass (Shizuoka, Japan).
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