D pbs

For isolation of sEVs, 6.0 × 10⁶ PANC-1 cells were seeded in thirteen 150 mm dishes and precultured with RPMI 1640 containing FBS and antibiotics for 24 h. They were washed twice with Dulbecco’s phosphate-buffered saline (D-PBS, Nacalai Tesque). As FBS also includes large amounts of sEVs, it is necessary to change to advanced RPMI 1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) containing 2 mmol/L glutamine (Thermo Fisher Scientific) and antibiotics. Thereafter, the cells were cultured for 48 h under hypoxia (1% O₂) or normal oxygen conditions (n = 3). Fresh (non-cultured) advanced RPMI 1640 medium was used as a blank (n = 3). Cell-conditioned medium was collected and centrifuged at 2000× g for 25 min at 4 °C to pellet and remove cells, debris, and apoptotic bodies. The supernatant was filtered using a 0.22 μm pore PES filter (Merck Millipore, Burlington, MA, USA) to remove large sEVs. The filtrate was concentrated with a 100 kDa cut-off filter (Merck Millipore). The concentrate was ultracentrifuged at 37,000 rpm (average RCF is 234,700× g) for 70 min at 4 °C (SW41Ti rotor, Beckman Coulter, Brea, CA, USA). The pellet was washed with physiological saline (Hikari Pharmaceutical, Tokyo, Japan) and was collected by ultracentrifuge. This washing procedure was repeated twice. Finally, the weight of the pellet was adjusted to 0.05 g (≈ 50 μL) by adding physiological saline.

FN labeled with rhodamine (20 μg, Bovine plasma, Cytoskelton Inc., CO, USA) was dissolved in Dulbecco's phosphate buffered saline (D-PBS, Mg²⁺ and Ca²⁺ free) (Nacalai Tesque, Kyoto, Japan) and the concentration of the fluorescent FN was adjusted to 8 μg/ml for subsequent FN adsorption experiments. The FN solution (800 μl) was added to three wells of each PDMS, Lar- and Low-PI-PDMS chambers with unstretched and stretched states followed by incubation at 37 °C or 20 °C for 20 h (n = 3). After incubation, FN solution (150 μl) was taken from the well followed by added to a well in a 96-well microplate (bottom clear type (655096)), Greiner Bio-One International GmbH, Kuremensmünster, Australia) (n = 2). The fluorescent intensity of the FN solution was measured using a plate reader (SpectraMax M2, Molecular Devices, LLC., CA, USA)). A filter for 540 nm excitation and 580 nm emission was used for the detection of the rhodamine-labeled FN. The amount of adsorbed FN was calculated from the difference in the fluorescence intensity of FN solutions before and after the incubation. The amount of adsorbed FN is shown as the mean value with standard deviation.

Cryopreserved tissues were suspended in CelLytic M (Sigma-Aldrich, MO, USA) and finely chopped using scissors. Thereafter, the chopped tissues were homogenised using an ultrasonic homogeniser on ice. The lysed tissues were centrifuged (14,000 × g for 10 min at 4 °C) to pellet cellular debris. The supernatant was then harvested and assessed for protein concentration using a BCA protein assay kit (Pierce, MA, USA), in accordance with the manufacturer’s instructions. Subsequently, the supernatant was diluted to 1 mg/mL with D-PBS (Nacalai Tesque).

BMDCs were cultured in 96-well plates at 1 × 10⁵ cells per well and either treated with 1 μg/ml LPS (InvivoGen), or transfected with 1 μg/ml LPS using 0.3% v/v FuGENE HD (Promega) after priming with 1 μg/ml Pam3CSK4 (InvivoGen) for 6 h. At 16 h after LPS stimulation, the IL-1β concentrations in the culture supernatants were measured using a Cytometric Bead Array (BD Biosciences) in accordance with the manufacturer’s instructions. As for the measurement of IL-1β in the synovium, synovial tissues were resected from the inflamed joints, cut into small pieces, and the tissue weight was measured. The resultant synovial tissues were placed into 500 μl of D-PBS (−) (Nacalai Tesque), then vortexed and centrifuged. The collected supernatants were then used for the measurement of the IL-1β concentration, which was performed using a Cytometric Bead Array, and the results were normalized with tissue weights of individual samples.

To monitor the expression of Cas13a mRNA and crRNA–guide RNAs, 2.5 × 10³ HEK293T cells were seeded per well; transfections were performed on the following day. Total RNA was extracted after 12, 24, 48, 72, and 96 h, and subjected to cDNA synthesis and RT-PCR. For half-life measurements, 6 × 10³ HEK293T cells were seeded per well. The next day, 375 ng each of Cas13a and crRNA–guide RNA vector were transfected separately. Culture media was replaced the following day. On the third day, for time point 0 (t0), cells were washed with D-PBS (Nacalai Tesque), lysed with 100 µL TRIzol reagent (Invitrogen), and then kept in a −80 °C freezer. For the remaining time points (2.5, 5, 7.5, 10, and 15 h), cells were simultaneously treated with 5 µg/mL ActD (final concentration). Total RNA was isolated from the cells and subjected to cDNA synthesis and qRT-PCR. The C_T value was normalized against human 18S rRNA and the corresponding value at t0. Half-life was estimated from t0-normalized data.
The half-life of a transcript was calculated as follows: half-life = ln(2)/kdecay [37 (link)]. This equation could be fit to the data using nonlinear regression (exponential one-phase decay; least square), which is available in GraphPad Prism.

The human esophagogastric junction cancer cell line (OE-19) was purchased from Summit Pharmaceuticals International Corporation (Tokyo, Japan). The cells were subcultured in an RPMI 1640 (Nacalai Tesque Co., Kyoto, Japan) medium adjusted so that the antibiotic–antimycotic solution (Life Technologies Co., CA, USA) and FBS were 1% and 10%, respectively. OE-19 cells were then seeded in a black-walled 96-well plate (Thermo Scientific Nunc; Thermo Fisher Scientific, MA, USA) at 1 × 10⁴ cells/well. After 24 h, the specimens were washed with D-PBS (Nacalai Tesque Co., Kyoto, Japan) and then with modified EPM2 (AthenaES, MD, USA). Next, specimens were switched to a modified EPM2 medium with each concentration of TCA (Sigma, MO, USA) and cultured at 37 °C with 5% CO₂. The added TCA concentrations were 0, 50, 100, 500, and 1000 μM. 48 h after adding the reagent, washing was performed twice with D-PBS. Then, D-PBS was added at 50 μL/well. Then, 4 μM Calcein-AM (Dojindo Laboratories, Kumamoto, Japan) was added to this at 50 μL/well and cultured for 1 h at 37 °C. The plate was removed and fluorescence intensity at 515 nm was measured with excitation rays at 490 nm.

HT29-CD63-Nluc cells (3.0 ×10⁶) were seeded into 150-mm dishes and pre-cultured with RPMI1640 containing 10% FBS and antibiotics for 24 h. The cells were washed twice with Dulbecco’s phosphate-buffered saline (D-PBS, Nacalai Tesque). Next, the culture medium was exchanged for advanced RPMI1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) containing 1 μmol/L glutamine (Thermo Fisher Scientific) and antibiotics and cultured for 2 days.
Equal numbers of cells (4.0 ×10⁶) of HCT116 wild-type (WT) cell and HCT116 IDH1 (R132H/+) cell were seeded into 150-mm dishes and pre-cultured with DMEM high glucose medium containing 10% FBS, antibiotics, and sodium pyruvate for 24 h. The cells were then washed twice with D-PBS. Next, the culture medium was exchanged for DMEM high glucose medium with 2% (v/v) exosome depleted FBS (Thermo Fisher Scientific) and antibiotics and cultured for 2 days before the cell-conditioned medium was collected. The cell-culture medium was centrifuged at 2,000 × g for 25 min and 15,000 × g for 50 min at 4°C to pellet and remove cells, debris, and apoptotic bodies. The supernatants were filtered using a 220-nm polyethersulfone filter (Merck Millipore Ltd., Burlington, MA, USA) to remove the large EVs. The filtrates were concentrated using a 100 kDa cut-off filter (Merck Millipore Ltd.). This suspension was used to prepare the samples.