389-Watabe-PGE2discharge/Fig3AB_PKA_ctrl_13
Author reviewedTime-lapse FRET images of reporter HeLa cells and producer HeLa cells
Project
Dataset ID
Fig3AB_PKA_ctrl_13
Kind
Image data
Title
Time-lapse FRET images of reporter HeLa cells and producer HeLa cells
Description
Time-lapse FRET images of Booster-PKA in reporter COX-DKO (COX-1 and COX-2 deficient)-HeLa cells cocultured with producer HeLa cells. In the producer cells, optogenetic calcium channel stimulator: OptoSTIM1 (CRY2clust) is expressed, and PGE2 (prostaglandin E2) is generated after blue light flash. It is released and binds to the EP2 on the reporter cells and induces the FRET of PKA biosensor, Booster-PKA. These serial processes reveal the coverage of PGE2 around one activated cell.
This dataset includs 5 files of serial imaging. The images of iRFP were obtained at the start and the end of the imaging showing the producer cells.
Folder Name
Fig3AB_PKA_ctrl_13
File Format
.nd2
File Size
580MB
File Separation Method
5 files of serial imaging
Figure Number
Fig3AB
Dimensions
2132x2132x1x2x25
X Scale
2.7 micrometer
Y Scale
2.7 micrometer
Z Scale
-
T Scale
1 minute
Channel
2 channel
Quantitative Data
-
Biosample
Description
Co-culture of the producer cells and the reporter cells.
Producer cells; HeLa cells expressing OptoSTIM, R-GECO1 and iRFP670.
Reporter cells; COX-1 and COX-2 (COX-DKO)-deficient HeLa cells which express Gs-coupled EP2 receptor and Booster-PKA
Organism
Homo sapiens (NCBITaxon:9606)
Strain
-
Cell
-
Cell line
HeLa cell (CLO:0003684)
MeSH
Intrinsic Variable
In the reporter cells, cyclooxygenases (COX1 and COX2) were knocked out by CRISPR/Cas9 to eliminate PGE2 synthesis.
Extrinsic Variable
OptoSTIM, R-GECO1 and iRFP670 were expressed in producer cells, and EP2 receptor and Booster-PKA were expressed in reporter cells.
Ontology
Anatomical Entity
-
UBERON
-
Biological Process
cell communication (GO:0007154)
signal transduction (GO:0007165)
Cellular Component
cytoplasm (GO:0005737)
Molecular Function
-
MeSH Anatomy
MeSH BP
MeSH CC
MeSH MF
Genetics
Gene
-
Protein / Probe
-Booster-PKA
Method / Genotype
transposition
Gene
PTGS1,
PTGS2
Protein / Probe
-
Method / Genotype
gene knock out
Gene
-
Protein / Probe
Prostaglandin E2 receptor EP2 subtype
Method / Genotype
transposition
Gene
-
Protein / Probe
-OptoSTIM1 (CRY113clust)
Method / Genotype
transposition
Gene
-
Protein / Probe
-R-GECO1
Method / Genotype
lentivirus infection
Gene
-
Protein / Probe
-
Method / Genotype
lentivirus infection
Treatments
Reagent / Compound
-
Concentration
-
Fold Dilution
-
Imaging Method
Imaging Method
Detection Method
wide-field detection
Visualization Method
Booster-PKA, a PKA biosensor,
mKate2
Illumination Method
widefield illumination
Source of Contrast
-
Imaged Parameter
fluorescence emission
Sample Preparation Method
living tissue
MeSH
Instrument
Body
Nikon ECLIPSE Ti2
Module
-
Light Source
Excelitas Technologies Corp. X-Cite TURBO LED light source+O4:AN4
Detector
Hamamartsu ORCA
Fusion Digital CMOS camera
Objective
Nikon CFI Plan 4X
Filter Set
A 555BP10 excitation filter (Omega Optical, Brattleboro, VT, USA), an FF562Di03 dichroic mirror (Semrock, Rochester, NY, USA), and XF3024 (590DF35) (Omega Optical) and BLP01-633R-25 (Semrock) emission filters for mKOκ and mKate2, respectively; for R-GECO, the same filter set as mKOκ; for iRFP670 imaging, an FF01-640/14 excitation filter (Semrock), a dichroic mirror 660(Nikon), and a 700/75 emission filter (Nikon).
Dichroic
see Filter Set