Planapon

The setup of the FLIM system consisted of a picosecond diode laser (laser power, 5 mW) with an emission wavelength of 470 nm (LDH470; PicoQuant, Germany) and a ~ 70 ps pulse width for the excitation of o-BMVC under a scanning microscope (IX-71 and FV-300; Olympus, Japan). The fluorescent signal from o-BMVC was collected using a 60 × NA = 1.42 oil-immersion objective (PlanApoN; Olympus, Japan), passed through a 550/88 nm bandpass filter (Semrock, USA), and detected using a single-photon avalanche diode (SPAD) (PD-100-CTC; Micro Photon Devices, Italy). The fluorescence lifetime was recorded and analyzed using a time-correlated single-photon counting (TCSPC) module and software (PicoHarp 300 and SymPhoTime v5.3.2; PicoQuant, Germany). FLIM images were constructed from pixel-by-pixel lifetime information.

Images were acquired on an inverted microscope (IX81, Olympus) with a heated incubator that maintained the chamber at 37 °C and 5% CO₂ (WELS, Tokai-Hit). Images were acquired for either 15 or 25 minutes at 1-minute intervals. Drugs were added at the 5-minute and 15-minute point. PKA activation was achieved through treatment with 50 µM forskolin and 100 µM IBMX, unless otherwise noted. For the experiment demonstrating reversibility, the media was replaced with new media containing 40 µM H89 at the 15-minute point, as residual forskolin and IBMX led to an incomplete inhibition of PKA. Images were acquired with a CMOS camera (C11440, Hamamatsu) in a 60x oil objective (Plan Apo N, Olympus) with the appropriate excitation and emission filters driven by a filter wheel controller. Metamorph was used to control the hardware associated with the microscope which includes the motorized stage (MS-2000, Applied Scientific Instrumentation), filter wheels (Lambda 10-3, Sutter Instruments), and LED light source (pE-300, CoolLED). To measure response with the FRET reporter, cells were excited in the CFP channel, and images were acquired in the YFP channel.

The setup of the FLIM system consisted of a picosecond diode laser (laser power, 5 mW) with an emission wavelength of 470 nm (LDH470; PicoQuant, Germany) and a ~70 ps pulse width for the excitation of o-BMVC under a scanning microscope (IX-71 and FV-300; Olympus, Japan). The fluorescent signal from of o-BMVC was collected using a 60× NA = 1.42 oil-immersion objective (PlanApoN; Olympus, Japan), passing through a 550/88 nm bandpass filter (Semrock, USA), followed by detection using a SPAD (PD-100-CTC; Micro Photon Devices, Italy). The fluorescence lifetime was recorded and analyzed using a time-correlated single-photon counting (TCSPC) module and software (PicoHarp 300 and SymPhoTime v5.3.2; PicoQuant, Germany). FLIM images were constructed from pixel-by-pixel lifetime information.
For the study of fixed cells, cells on coverslip were fixed with 70% ethanol for 10 min and then stained with 5 µM o-BMVC for 10 min at room temperature. For the study of PDS pretreatment, HeLa and MRC-5 cells on coverslip were treated with 1 µM PDS overnight. After washing twice, cells were fixed with 70% ethanol for 10 min and then stained with 5 µM o-BMVC for 10 min at room temperature. For the study of DNase treatment, HeLa cells on coverslips were fixed with 70% ethanol for 10 min and then treated with 20 µg/ml DNase for 1 h at 37 °C followed by 5 µM o-BMVC staining for 10 min.