Spinning disk confocal microscope

Alexa Fluor 488 was conjugated onto the C terminus of pyoS2^NTD as described in Supporting Information. P. aeruginosa PAO1 cells were grown in M9-glucose media (6.78 g/L Na₂HPO₄, 3 g/L KH₂PO₄, 0.5 g/L NaCl, 10 mM d-glucose, 1 mg/mL NH₄Cl, 2 mM MgSO₄) at 37 °C, and were labeled with 1 μM fluorophore-conjugated pyoS2 construct for 15 min (details are provided in Supporting Information). FRAP experiments were performed using a PerkinElmer spinning disk confocal microscope with a 100× oil-immersion objective (1.4 N.A.). Images were acquired using the 488-nm laser at 10% power, and bleaching was performed at 50% laser power at maximum speed. Recovery images were acquired over a time course up to 2 min. Bright-field images were recorded for each FRAP experiment.

Transfected cells (Jurkat or Reh) were seeded onto poly-L-lysine (cat. no. P8120; Beijing Solarbio Science & Technology Co., Ltd.) coated glass slides (20,000 cells/slide). Cells were fixed with 4% paraformaldehyde (Sigma-Aldrich; Merck KGaA) for 15 min at room temperature, washed three times with PBS for 5 min, permeabilized with 0.1% Triton X-100 for 10 min and blocked with 2% BSA (cat. no. 0332; Amresco, LLC) for 1 h at room temperature. Subsequently, cells were incubated with primary antibodies against CREBBP (cat. no. sc-369; 1:500; Santa Cruz Biotechnology, Inc.) and E2F3 (cat. no. sc-56665; 1:500; Santa Cruz Biotechnology, Inc.) overnight at 4°C. Following the primary incubation, cells were incubated with goat anti-mouse (1:2,000; cat. no. A32727) and goat anti-rabbit (1:2,000; cat. no. A32731; both from Thermo Fisher Scientific, Inc.) secondary antibodies at room temperature for 1 h. The nuclei were stained with DAPI (100 ng/ml; cat. no. ZLI-9557; OriGene Technologies, Inc.) at room temperature for 15 min and fluorescence images were observed under a spinning disk confocal microscope (PerkinElmer, Inc.), with a ×100 oil-immersion objective lens.