Sw 32 ti swinging bucket rotor

Membrane fraction was prepared, as described previously [30 (link)]. V5-tagged Rab35 wild-type, T75A, or T75D mutants were transfected with Lipofectamine 2000 into HEK-293. Cells were washed once with PBS and scraped in lysis buffer containing 0.25 M sucrose, 1 mM EDTA, 10 mM Hepes-NaOH (pH 7.5), 1 mM MgCl₂ and protease inhibitors. Cell lysates were passed through a 27 gauge needle 10 times and centrifuged at 1000 g for 15 min at 4 °C. Protein levels were quantified using the BCA kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal amount of protein from each sample was loaded to centrifuge tubes (Beckman Coulter, Indianapolis, IN, USA) and centrifuged using an SW 32 Ti swinging bucket rotor (Beckman Coulter, Indianapolis, IN, USA) for 1 h at 100,000 g at 4 °C. The pellets were rinsed with lysis buffer and centrifuged for 1 h at 100,000 g at 4 °C. Pellets were resuspended in Laemmini sample buffer and subjected to SDS-PAGE. Western blot analysis was performed with anti-V5, anti-caveolin-1 (1:1000, 3267, Cell Signaling, Danvers, MA, USA), anti-EEA1 (1:1000, 3288, Cell Signaling, Danvers, MA, USA), anti-GOPC (1:1000, 8576, Cell Signaling, Danvers, MA, USA), and anti-Hsp90 (1:1000, ab13492, Abcam, Cambridge, UK) antibodies.

Cells were grown in the presence of serum until they reached a subconfluent state (80%–90%) at 37°C and 5% CO₂. Then, they were gently rinsed three times with phosphate-buffered saline (PBS) and cultured with serum-free DMEM/F12 for 48 h. The cell-conditioned medium was collected and subjected to gradient centrifugation (300 × g for 10 min, 2,000 × g for 10 min, and 10,000 × g for 30 min) to remove residual cells and debris. Exosomes were pelleted from the supernatants through ultracentrifugation at 100,000 × g for 70 min by using an SW32 Ti swinging bucket rotor (Beckman Coulter, Fullerton, CA, USA). The pellets were resuspended in PBS, pooled, and ultracentrifuged once more at the same high speed for 70 min to eliminate contaminating proteins. The final exosome pellet was resuspended in PBS for further experiments.

Exosomes were isolated following a previously described ultracentrifugation protocol [15 (link)]. After incubation for 72 h at 37°C with 5% CO₂, cells were centrifuged at 300×g for 5 min. The supernatant was then passed through a 0.22-μm filter. This filtered supernatant was transferred to a fresh tube (50 mL) and centrifuged at 2,000×g for 30 min. The supernatant obtained from this procedure was then transferred to ultracentrifuge tubes and spun in a SW32Ti swinging bucket rotor (Beckman Coulter, Brea, CA, USA) at 12,000×g for 30 min at 4°C. The supernatant was again transferred to new ultracentrifuge tubes and spun for 70 min at 110,000×g. The supernatant was then discarded and the pellet was suspended in 1 ml of sterile phosphate-buffered saline (PBS). Samples were then transferred to 1.5-ml microtubes and supplemented with 200 μL of ExoQuick-TC (System Biosciences, Mountain View, CA, USA). After incubation at 4°C overnight, the mixture was centrifuged at 1,500×g for 30 min. The supernatant was then discarded and the pellet was centrifuged at 1,500×g for 5 min. Finally, the resulting pellet was suspended in 100 μL of sterile PBS.

Exosomes from serum and cell conditioned media were both isolated by ultracentrifugation method. Briefly, the serum was first centrifuged 15 min at 3,000 ×g, 4°C, followed by 30 min, 10,000 ×g. Then, the supernatant was transferred into a 6-mL ultracentrifuge tube (Beckman, 344619). The tube was then filled with PBS (HyClone, SH30256.01) and ultracentrifugation was done twice for 70 min at 100,000 ×g, 4°C in a Type 100 Ti swinging-bucket rotor (Beckman). As for conditioned media, it was first centrifuged for 5 min at 500 ×g, 4 ×g, followed by for 30 min at 2,000 ×g, and for 35 min at 10,000 ×g. Then, the supernatant was transferred into 38.5 mL ultracentrifuge tubes (Beckman, 326823) and ultracentrifuged twice for 70 min at 100,000 ×g, 4°C, in an SW 32 Ti swinging-bucket rotor (Beckman). The deposits in each step are, respectively, referred to as intact cells in suspension during cell culture, cell debris, extracellular vesicles (EVs), and exosomes as shown in Fig. S5A. For exosomal TAZ level comparison, a filtration process by a 0.22 μm filter was performed to exclude particles >200 nm before ultracentrifugation (Lobb et al., 2015 ; Jeppesen et al., 2019 (link)).

FBS was ultracentrifugated at 120,000 × g for 10 h to remove EVs and then added into DMEM at final concentration of 10% (v/v). HEK293 and MC38 cells were cultured in this DMEM until approximate 90% confluence in 10‐cm cell culture dishes. Then, the conditioned media were collected and subjected to a centrifugation step at 300 × g and room temperature (RT) for 10 min to pellet and remove the cells. All subsequent centrifugation steps were performed at 4°C. Next, the supernatant was spun at 2,000 × g for 20 min to remove debris and apoptotic bodies and then centrifuged at 10,000 × g for 30 min to remove large EVs. Then, the media supernatants from the centrifugation step at 10,000 × g were passed through 0.22 μm PVDF filters (Millipore) and subjected to ultracentrifugation at 120,000 × g for 70 min by Beckman XPN‐100 with an SW 32 Ti swinging bucket rotor (Beckman Coulter) to sediment EVs. The crude EV pellets were resuspended in a large volume of ice‐cold PBS, followed by ultracentrifugation at 120,000 × g for 70 min to wash the samples. The final pellets were resuspended in ice‐cold PBS. The concentrations of exosomal proteins were measured using a BCA Protein Assay Kit (Thermo Fisher) according to the manufacturer's instructions.