Abstract
Methods Plasmids encoding GFP-tagged wild type and D842V mutant PDGFRa were expressed in hTERT-RPE1 cells. To promote ciliogenesis, transfected cells were grown under serum-free conditions for 12 hours and in the presence of various inhibitors. Cilia numbers of GFPpositive cells were assessed by immunofluorescence microscopy with antibodies against cilia markers. In parallel, cells were analyzed by western blotting to evaluate expression level and activity of the tagged receptors.
Highlights
The receptor tyrosine kinase (RTK) Platelet-Derived Growth Factor Receptor a (PDGFRa) localizes to the primary cilium, and expression of the constitutive active PDGFRa D842V mutant has been linked to gastrointestinal tumor formation
Our results indicate that the D842V mutant promotes loss of primary cilia by a mechanism that depends on its kinase activity, but not on activation of AKT, ERK1/2 or HDAC6
Cilia numbers of D842V-expressing cells were restored to control levels upon treatment with the RTK inhibitor crenolanib, but not with other RTK inhibitors such as AG1296 or imatinib
Summary
The receptor tyrosine kinase (RTK) Platelet-Derived Growth Factor Receptor a (PDGFRa) localizes to the primary cilium, and expression of the constitutive active PDGFRa D842V mutant has been linked to gastrointestinal tumor formation. We set out to investigate whether expression of D842V causes loss of primary cilia in cultured cells
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