High-content screening assay for activators of the Wnt/Fzd pathway in primary human cells.
We have developed a high-content screening (HCS) assay to find activators of Wnt/Frizzled (Wnt/Fzd), a pathway known to be important in bone formation. Utilizing primary human preosteoblasts as a model, activation of the Wnt/Fzd pathway was detected by monitoring the stabilization and translocation of the transcription factor beta-catenin from cytoplasm to the nucleus. Endogenous beta-catenin was detected in preosteoblasts by immunofluorescent staining, and subcellular localization was determined by HCS using the Cellomics (Pittsburgh, PA) ArrayScan IV. Positive controls, including Wnt3A-conditioned medium and inhibitors of glycogen synthase kinase-3beta, resulted in increased nuclear beta-catenin. The assay had a Z'-factor of 0.6 and was conducive to automation for high-throughput screening/HCS. By combining standard immunofluorescence technology with automated fluorescence microscopy, we demonstrate the capability of screening cell-signaling pathways in primary human cells.
Borchert
Kristen M
KM
Lilly Research Laboratories, Eli Lilly & Co., Research Triangle Park, NC, USA.
Galvin
Rachelle J Sells
RJ
Frolik
Charles A
CA
Hale
Laura V
LV
Halladay
David L
DL
Gonyier
Robin J
RJ
Trask
O Joseph
OJ
Nickischer
Debra R
DR
Houck
Keith A
KA
D001681:Biological Assay / Q000379:methods*; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D019085:Fluorescent Antibody Technique, Direct / Q000379:methods; D051157:Frizzled Receptors; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D004364:Pharmaceutical Preparations; D011506:Proteins / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects* / Q000502:physiology*; D015534:Trans-Activators / Q000378:metabolism*; D051153:Wnt Proteins; D051176:beta Catenin
C495270:CTNNB1 protein, human; D003598:Cytoskeletal Proteins; D051157:Frizzled Receptors; D036341:Intercellular Signaling Peptides and Proteins; D004364:Pharmaceutical Preparations; D011506:Proteins; D015534:Trans-Activators; D051153:Wnt Proteins; D051176:beta Catenin