Mechanical stress activates the JNK pathway. S2R+ cells transiently transfected with dJun-FRET biosensor (A) or the controls mCFP alone (B) and mCFP-dJun (C) were plated on collagen-coated silicone membranes on a Stage Flexer set up. Donor mCFP FL was collected before stretching (black) and at different times after continuous static stretch (1 hour-red in A, 2 hours-blue in A, and 3 hours-green in A and red in B and C). dJun-FRET sensor activity increased upon stretching, reaching a maximum after 2 hours. FL of controls was not affected. Donor mCFP FL from a specific region of interest containing ∼15–30 cells were collected after 3 hours of mechanical stretch (D). Thereafter, the acceptor mYFP was photobleached (95%) and donor mCFP FLs were re-collected (E). Panel F shows the donor mCFP FL histograms for the dJun-FRET biosensor before (black) and after (red) acceptor (mYFP) photobleaching. Altogether, these data showed that FL changes observed after mechanical stretch can be attributed directly to energy transfer between the donor and acceptor fluorophores in the dJun-FRET biosensor.
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