Visiview 4

All proteins (p53¹⁻⁹⁴, p53^TAD2, p53^DBD, and p53¹⁻³¹²) and PR25/GR25 were prepared in 20 mM Hepes pH 7.0, 50 mM NaCl, 2 mM TCEP, and 0.04% (w/v) NaN_3. The 30 μL sample was plated on a 30 mm No. 1 round glass coverslip and mounted on an Observer D1 microscope with 100×/1.45 oil immersion objective (Zeiss, Oberkochen, Germany). Protein droplets were viewed using HAL 100 halogen lamp, and images were captured with an OrcaD2 camera (Hamamatsu, Shizuoka, Japan) using VisiView 4.0.0.13 software (Visitron Systems GmbH, Puchheim, Germany). Droplet formation was induced by the addition of PR25/GR25 in a fixed concentration of protein. Images were taken every 5 min until 30 min after the addition of peptides. Microscopy images were processed using Fiji/ImageJ 1.53a software (Bethesda, US), applying linear enhancement for brightness and contrast.

All live-cell microscopy experiments were performed on an Olympus IX73 inverted microscope if not mentioned otherwise. The microscope is equipped with an UApoN340 40× oil immersion objective (Olympus, Tokyo, Japan) and a CCD Retiga R1 camera (Q-imaging, Tucson, AZ, Canada). For illumination, a LedHUB^® (Omicron, Vienna, Germany) equipped with 340, 385, 455, 470, and 550 nm LEDs in combination with CFP/YFP/RFP (CFP/YFP/mCherry-3X, Semrock, Rochester, NY, USA) or GFP (GFP-3035D, Semrock, Rochester, NY, USA) filter set was used. Alternatively, an AnglerFish F-G/O (NGFI, Grraz, Austria) has been used. Data acquisition and control of the fluorescence microscope were performed using Visiview 4.2.01 (Visitron, Puchheim, Germany).

All live-cell microscopy experiments were performed on an Olympus IX73 inverted microscope if not mentioned otherwise. The microscope is equipped with an UApoN340 40× oil immersion objective (Olympus, Japan) and a CCD Retiga R1 camera (Q-imaging, Canada). For illumination, a LedHUB® (Omnicron, Germany) equipped with 340, 385, 455, 470, and 550 nm LEDs in combination with CFP/YFP/RFP (CFP/YFP/mCherry-3X, Semrock, USA) or GFP (GFP-3035D, Semrock, USA) filter set was used. During the measurements cells were continuously perfused by a gravity-based perfusion system (NGFI, Graz, Austria). Data acquisition and control of the fluorescence microscope was performed using Visiview 4.2.01 (Visitron, Germany).

Experiments were performed with an Olympus IX73 inverted microscope that is equipped with an UApoN340 40× oil immersion objective (Olympus, Tokyo, Japan) and a CCD Retiga R1 camera (Q-imaging, Surrey, BC, Canada). For illumination, LedHUB^® (Omnicron, Germany) equipped with 340, 385, 455, 470, and 550 nm LEDs in combination with CFP/YFP/RFP (CFP/YFP/mCherry-3X, Semrock, New York, NY, USA) filter set was used. Visiview 4.2.01 (Visitron, Puchheim, Germany) was used for the data acquisition. Alternatively, an AnglerFish F-G/O (Next Generation Fluorescence Imaging/NGFI (www.ngfi.eu, accessed on 16 May 2022), Graz, Austria) was used for data acquisition. During the measurements, cells were perfused by a gravity-based perfusion system PS-9D (NGFI, Graz, Austria). Briefly, nine positions of the valve are connected with reservoirs and the reservoir of interest can be automatically activated via perfusion control software. The flow rate of the reservoir manually adjusted to 1 ml/min. All the experiments were done at room temperature without specific temperature or gas control. Cells were chosen randomly based on the expression of respective genetically encoded biosensors, and in the case of mitochondrial-targeted probes, based on correct localization.

If not stated otherwise, all experiments were performed with a Zeiss array confocal laser scanning microscope (Axio Observer.Z1 from Zeiss, Gottingen, Germany) by using 100x objective lens (Plan-Fluor x100/1.45 Oil, Zeiss, Germany). This was equipped with a motorized filter wheel (CSUX1FW, Yokogawa Electric Corporation, Tokyo, Japan) on the emission side, an AOTF-based laser merge module for the 405, 445, 473, 488, 514, and 561 nm laser lines (Visitron Systems), and a Nipkow-based confocal scanning unit (CSU-X1, Yokogawa Electric Corporation). Data acquisition and control of the fluorescence microscope were performed using Visiview 4.2.01 (Visitron, Puchheim, Germany).

Cytosolic Ca²⁺ measurement without UV light was performed with an Olympus IX73 inverted microscope. This microscope was equipped with an UApoN340 40× oil immersion objective (Olympus, Tokyo, Japan) and a CCD Retiga R1 camera (Q-imaging, Vancouver, BC, Canada). LedHUB^® (Omnicron, Rodgau, Germany) equipped with 340, 385, 455, 470, and 550 nm LEDs. A GFP (GFP-3035D, Semrock, Henrietta, NY, USA) filter set was used for illumination of Fluo-4. Visiview 4.2.01 (Visitron, Puchheim, Germany) was used for the data acquisition. Alternatively, an AnglerFish F-G/O (Next Generation Fluorescence Imaging/NGFI (www.ngfi.eu), Graz, Austria) was used for data acquisition. Subsequent data analysis was performed in ImageJ (NIH, Bethesda, MD, USA) and Excel (Microsoft, Redmond, WA, USA). Both microscopes were equipped with an automatic perfusion system PS-9D (NGFI).
To avoid usage of UV light, cells were loaded with 3.3 µM Fluo-4 in EB for 30 min. On the microscope, cells were perfused with 2 mM Ca²⁺ buffer for 24 min and 2-min 30 mM K⁺ buffer to get maximum uptake through the L-type Ca²⁺ channels. The depolarization induced maximum Ca²⁺ uptake via L-type Ca²⁺ channel is used for the normalization of Fura-2. The number of cytosolic Ca²⁺ spikes was calculated from min 2 to min 24.