Integrin-Dependent Activation of the JNK Signaling Pathway by Mechanical Stress
Figure 1
FRET-FLIM quantification of dJun-FRET biosensor is a readout of the activity of the JNK pathway in response to chemical agonists and antagonists.
S2R+ cells were transiently transfected with dJun-FRET (A) biosensor, and fluorescence lifetimes (FL) of mCFP were collected 48 hours post transfection. Cells were left untreated (black) or subjected to treatment with LPS, a JNK signaling activator (red) or L-JNKI1, a JNK inhibitor (blue) for 2 hours before FLIM measurements. Curves represent FLIM data recorded from ∼75 cells for each condition. The chemical activator and inhibitor modulated the donor FL of dJun-FRET, while no effect was observed on the controls. A direct measurement of sensor activity on S2R+ cells plated on plastic was performed. Untreated cells (B) or those treated with LPS (C) or L-JNKI1 (D) were stained with anti-Phospho-c-Jun antibody and phalloidin-TRITC. P-Jun staining was quantified by calculation of the average integrated density (the product of Area and Mean Gray Value) of ∼100 cells (nucleus and cytoplasm) (E). LPS treatment led to morphological changes in S2R+ cells (from a pseudopolygonal flat shape to a filopodia-rich compacted aspect) and a statistically significant (p<5×10−7) increment of p-Jun staining (cytoplasm). L-JNKI1 treatment yielded cells with numerous multibranched thick filopodia and a statistically significant (p<5×10−5) decrease in P-Jun levels (cytoplasm).