CIL:26534, Mus musculus, fibroblast
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Lee, Chan-Soo; Choi, Chang-Ki; Shin, Eun-Young; Schwartz, Martin Alexander; Kim, Eung-Gook (2021). CIL:26534, Mus musculus, fibroblast. In Cell Image Library. UC San Diego Library Digital Collections. Dataset. https://doi.org/10.6075/J0BC3X9P
- Description
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To study the molecular mechanism by which nonmuscle myosin II (MII) regulates protrusion and adhesion dynamics in migrating cells, NIH3T3 cells were transfected with myc-tagged phosphomimetic constitutively active MLC mutants (MLCaa, green). After 20' exposure to PDGF, which induces myosin-inactivation, stress fiber disassembly, and stimulates fibroblast motility, cells were co-stained for βPIX (a Rac1/Cdc42-specific GEF highly implicated in cell motility, red) and filamentous actin (blue). These findings help elucidate a functional link between MII and Rac1/Cdc42 GTPases, which may regulate protrusion/adhesion dynamics in migrating cells. This image is original data file Fig. 7C "PDGF-induced dissociation of the MII–βPIX complex," in J. Cell Biol. 2010. Vol. 190(4):663–674
- Date Issued
- 2021
- Researchers
- Methods
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Cells were cultured in DME (Invitrogen) supplemented with 10% fetal bovine serum and 100 U/ml penicillin/streptomycin (Invitrogen) at 37°C in a humidified 5% CO2 incubator. For transfections, cells in 60-mm-diameter dishes or on fibronectin-coated coverslips were incu- bated with a mixture of DNA and LipofectAMINE 2000 (Invitrogen) according to the manufacturer's instructions. The cDNA for MLC was subcloned into pCMV-myc (Takara Bio Inc.). Mutant constructs of MLC were generated using a QuikChange site-directed mutagenesis kit (Agilent Technologies) according to the manufacturer's protocol. In this myc-tagged MLC mutant, Ser1/Ser2 were replaced by alanines, and expressed in NIH3T3 cells.
In some experimental conditions, 16 h after replating onto fibronectin-coated coverslips, cells were treated with 50 ng/ml PDGF for 20 min. Cells were fixed 24–48 h after transfection using 3.7% paraformaldehyde in PBS for 15 min, permeabilized using 0.2% Triton X-100 in PBS for 2 min, blocked with 2% BSA in PBS, and co-stained for MLC (green), TRIO (red), and actin (blue). Images were captured by Zeiss LSM 710 confocal microscope with Plan-Apochromat 63X objective. - Technical Details
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Preparation: formaldehyde fixed tissue; detergent permeabilized
Relation to intact cell: dispersed cells in vitro
Item type: recorded image
Imaging mode: confocal microscopy
Parameter imaged: fluorescence emission
Source of contrast: distribution of epitope; distribution of a specific protein
Visualization methods: Alexa Fluor 488; Alexa Fluor 546; phalloidin
Data qualification: Raw - Series
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- Identifier
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Samplenumber: 26534
- Related Resources
- Source Record in the Cell Image Library: https://doi.org/10.7295/W9CIL26534
- J. Cell Biol. 2010. Vol. 190(4):663–674. https://www.ncbi.nlm.nih.gov/pubmed/?term=0713598
- BioStudies (previously JCB DataViewer): https://www.ebi.ac.uk/biostudies/studies/
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- License
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Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International Public License
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- UC Regents
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Under copyright (US)
Use: This work is available from the UC San Diego Library. This digital copy of the work is intended to support research, teaching, and private study.
Constraint(s) on Use: This work is protected by the U.S. Copyright Law (Title 17, U.S.C.). Use of this work beyond that allowed by "fair use" or any license applied to this work requires written permission of the copyright holder(s). Responsibility for obtaining permissions and any use and distribution of this work rests exclusively with the user and not the UC San Diego Library. Inquiries can be made to the UC San Diego Library program having custody of the work.
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Research Data Curation Program, UC San Diego, La Jolla, 92093-0175 (https://lib.ucsd.edu/rdcp)
- Last Modified
2022-08-12