Glass bottom

Vesicles isolated by precipitation from human serum were incubated with monoclonal anti-human FHR-1^{10 (link)} antibody conjugated with Alexa Fluor 647 (1:200) to detect FHR-1. FHR-1-transporting EVs were captured using beads coated with monoclonal FHR-1 antibody and were stained with Alexa Fluor 647-labeled anti-human CD9 antibody (1:100; Novus Biological, cat no. NB500-327). Samples were transferred into 35 mm culture dishes with a glass-bottom (Greiner). To detect nucleic acids in vesicles, the vesicles were incubated with 5 µM SYTOX orange (Thermo Fisher Scientific). Pictures were taken with LSM and analyzed using ZEN 2011 software.
PLA assays were performed to evaluate the co-localization of the vesicle markers CD9 and FHR-1. Vesicles isolated by precipitation from human serum were seeded onto 6.7 mm diagnostic slides coated with poly-l-lysine. The vesicles were fixed with 4% formaldehyde, blocked with Duolink blocking solution (Sigma-Aldrich), and incubated with mouse antibody to human FHR-1^{13 (link)} (1:200) and rabbit antibody to human CD9 (1:200; Abcam, cat no. ab92726). PLA assays were performed using the Duolink In Situ Red Starter Kit Mouse/Rabbit (Sigma-Aldrich, cat no. DUO92101) according to the manufacturer’s instructions. Images were captured with LSM 710 fitted with ZEN 2011 software.

After amino acid starvation and re-stimulation, cells were rinsed twice with ice-cold PBS and mounted on dishes with glass bottom (Greiner Bio-One, Stuttgat, Germany) using SmearGell (GenoStaff, Tokyo, Japan). The embedded samples were fixed with 4% paraformaldehyde/PBS for 15 min at room temperature. They were then rinsed twice with PBS, and permeabilized with 0.5% Triton X-100 in PBS for 6 min. After rinsing twice with PBS, the samples were blocked for 40 min with Blocking-One (Nacalai Tesque, Kyoto, Japan), and then incubated with primary antibodies in Blocking-One overnight at 4°C, rinsed twice with PBS, and incubated with Alexa Fluor 488 and 594-conjugated secondary antibodies (Abcam, Cambridge, UK) for 30 min at room temperature. The cells were washed with PBS and DAPI (Sigma-Aldrich) for nuclear staining, and then rinsed with 0.1% Triton X-100. Images were acquired on a spinning disk confocal super-resolution microscope (SpinSR10, Olympus, Tokyo, Japan) with a 100 X oil immersion objective lens (Olympus). To quantify co-localization, Pearson’s correlation coefficients were calculated from approximately 50–100 cells per sample using Cellsens imaging software (Olympus) with manually set thresholds. All the images were obtained and analyzed in a single setting. The primary antibodies used were mTOR (CST #2983; 1:400) and LAMP1 (Santa Cruz Biotechnology #SC-20011; 1:400).

Biomass was quantified by using a crystal violet assay, CFU counts were determined by plating, and cell viability was determined by a resazurin-based assay as described by Peeters et al. (33 (link)). For confocal laser scanning microscopy, biofilms were grown in a 96-well plate with a glass bottom (Greiner Bio-One), stained with a LIVE/DEAD solution (0.3% SYTO 9 and propidium iodide in PS; Life Technologies) for 15 min, and visualized with a motorized Nikon TE2000-E inverted microscope (Nikon Benelux) (36 (link)).

For fluorescence microscopy, proteins were visualized by fusion to GFP, 3 X mRFP or mCherry. The nuclear chromatin region was stained with bisbenzimide H33342 fluorochrome trihydrochroride (Hoechst 33342). Living cells were observed under a fluorescence microscope (model BX51; Olympus, Tokyo, Japan), and images were obtained by a BX51 fluorescence microscope (Olympus) equipped with an ORCA-R2 camera (Hamamatsu Photonics, Hamamatsu, Japan). Filter sets U-MWU (Olympus), U-MWIB (Olympus), and U-MWIG2 (Olympus) were used for Hoechst 33342, GFP and mCherry/3 X mRFP, respectively. Image acquisition and processing were carried out by using Aquacosmos (Hamamatsu Photonics) and Image J (National Institutes of Health, Bethesda, USA) software. Time-lapse observation was performed as follows. Cells were incubated on SSA at 28°C for 16 hours. After conjugation, cells were inoculated to SSA medium on a cell-culture dish with a glass bottom (Greiner Bio-One, Frickenhausen, Germany) and observed under a fluorescence microscope (model IX71; Olympus) as described in Nakamura et al. (2008) [31 (link)]. Images were processed with Image J.

Cells were seeded in 96-well plates with glass bottom (Greiner Bio-One, Frickenhausen, Germany) at a density of 10,000 cells per well and were allowed to attach overnight. The next day, cell nuclei were stained with Hoechst 33342 staining (1 mg/mL in H₂O; 1000× diluted). JetPEI/pDNA/AuNP 5 pt complexes were prepared as described above. Cells were incubated with JetPEI/pDNA/AuNP 5 pt complexes in Opti-MEM for 1 h at 37 °C. After washing the particles off, the cells were provided with full cell culture medium and live-cell imaging was performed using a confocal laser scanning microscope (C1si, Nikon, Tokyo, Japan). A Plan Apo VS 60× 1.4 NA oil immersion objective lens (Nikon, Tokyo, Japan) was used to obtain a pixel size of 70 nm and AuNP were detected by the reflected laser light of the 561 nm laser. Image processing was performed using ImageJ (FIJI) software.

Astrocytes were grown on cell culture dishes with a glass bottom (Greiner; 65,000 cells/cm²). The cells were loaded with Fluo-4 A.M. (4 μm, Invitrogen) for 20 min and then incubated for a further 30 min in 37°C before measurement. Imaging was performed in normal extracellular solution (NES) containing 136 mm NaCl, 2.5 mm KCl, 10 mm HEPES, 1.3 mm MgCl₂, 10 mm glucose, and 2 mm CaCl₂, pH 7.3 (Royle et al., 2008 (link)) as described previously (Terunuma et al., 2015 (link)). In some experiments, extracellular calcium was chelated with 2 mm EGTA added into the buffer. Fluorescence images were acquired with a Zeiss Cell Observer Spinning Disk Confocal microscope using epifluorescence illumination (excitation filter bandpass 470/20 nm, emission 525/50 nm), LD LCI Plan-Apochromat 25×/0.8W objective, Zen 2012 Blue software (Carl Zeiss) and Hamamatsu ORCA-R2 camera (Hamamatsu). Images were captured at 1-s intervals for up to 2 min (in some experiments up to 4 min) in 37°C and 5% CO₂. Image data were analyzed by Zen 2012 Blue software and subsequently by OriginPro 2016 software (OriginLab). The change in intracellular free calcium concentration ([Ca²⁺]_i) is represented by relative fluorescence intensity [(F₁ – F₀)/F₀, relative unit (r.u.)] (F₀, at rest; F₁, after administration of drugs, background subtracted) in the selected cytoplasmic or nuclear parts of the cells.

Wild-type HEK293T cells (2 × 10⁵) were seeded in a complete growth medium onto a 35 mm cell culture dish with a glass bottom (Greiner Bio-One, Kremsmünster, Austria). 20–24 h after plating the cells were transfected with a combination of plasmids bearing genes encoding the wild-type CST fused with either large (LgBiT) or small (SmBiT) subunit of NanoLuc luciferase (Table S5, 250 ng of each plasmid, 500 ng in total) using the FuGENE^® HD transfection reagent (Promega, Madison, WI, USA); 2–3 h before the measurement the conditioned medium was replaced with the serum-free OPTI-MEM medium (Life Technologies, Carlsbad, CA, USA). Immediately before the measurement, the Live Cell Reagent (Promega, Madison, WI, USA) was added to the cells according to a scaled-up protocol and the cells were immediately examined with an LV200 bioluminescence imaging system (Olympus, Tokyo, Japan).