389-Watabe-PGE2discharge/FigS1_MDCK_ctrl
Author reviewedTime-lapse FRET images of Booster-PKA in reporter COX-DKO-MDCK cells cocultured with producer MDCK cells
Project
Dataset ID
FigS1_MDCK_ctrl
Kind
Image data
Title
Time-lapse FRET images of Booster-PKA in reporter COX-DKO-MDCK cells cocultured with producer MDCK cells
Description
Time-lapse FRET images of Booster-PKA in reporter COX-DKO (COX-1 and COX-2 deficient)-MDCK cells cocultured with producer MDCK cells. In the producer cells, DREADD-Gq and GCaMP6s are expressed. After stimulation with DREADD-Gq ligand, PGE2 (prostaglandin E2) is generated in the producer cells. 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. In this dataset, the time-lapse images of GCaMP6s is also reposited.
Folder Name
FigS1_MDCK_ctrl
File Format
.nd2
File Size
159MB
File Separation Method
-
Figure Number
FigS1C
Dimensions
1152x1152x1x3x21
X Scale
-
Y Scale
-
Z Scale
-
T Scale
-
Channel
3 channels
Quantitative Data
-
Paper(s)
Biosample
Description
Co-culture of the producer cells and the reporter cells.
Producer cells; MDCK cells expressing DREADD-Gq and GCaMP6s.
Reporter cells; COX-1 and COX-2 (COX-DKO)-deficient MDCK cells which express Booster-PKA
Organism
Canis lupus familiaris (NCBITaxon:9615)
Strain
-
Cell
-
Cell line
MDCK cell (CLO:0007646)
MeSH
Intrinsic Variable
In the reporter cells, cyclooxygenases (COX1 and COX2) were knocked out by CRISPR/Cas9 to eliminate PGE2 synthesis.
Extrinsic Variable
DREADD-Gq and GCaMP6s were expressed in producer cells.
Booster-PKA were expressed in reporter cells.
DREADD-Gq ligand, Clozapine N-oxide was applied to stimulate the producer 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
-DREADD-Gq
Method / Genotype
transposition
Gene
-
Protein / Probe
-GCaMP6s
Method / Genotype
transposition
Treatments
Reagent / Compound
Clozapine N-oxide
Concentration
1 micromolar
Fold Dilution
-
Imaging Method
Imaging Method
Detection Method
wide-field detection
Visualization Method
Booster-PKA, a PKA biosensor,
mKate2,
GCaMP6s
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 10X
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 iRFP670 imaging, an FF01-640/14 excitation filter (Semrock), a dichroic mirror 660(Nikon), and a 700/75 emission filter (Nikon). for YC3.60, a 434/32 excitation filter (Nikon), a dichroic mirror455 (Nikon), and 480/40 and 535-30 emission filters (Nikon) for CFP and YFP, respectively
Dichroic
see Filter Set