0.2 m filter

The 60 × 10⁶ human melanoma cells were grown in serum-free MIM for 48 h. Collected supernatant fractions were filtered with a 0.2 µm filter (Sarstedt, Nümbrecht, Germany). Vesicles were pelleted by ultracentrifugation at 34800 rpm for 70 min at 4 °C (Beckman Coulter 55.2 Ti rotor, Krefeld, Germany). The pellet, containing vesicles, was resuspended in 100 µl PBS. Vesicle concentration and size were measured using the Nanosight technology equipped with a blue laser (405 nm) for real-time characterization of the vesicles.

Curcumin (purity 95.0%, C110685), tween-80, was purchased from Aladdin Chemical Reagents, Shanghai, China; TPGS was obtained from Professional Compounding Centers of America, Houston, TX, USA. Chloroform [high performance liquid chromatography (HPLC) grade] was provided by Scharlau, Barcelona, Spain and 0.2-µm filter was purchased from SARSTEDT AG & Co. KG, Nümbrecht, Germany. Methanol (analytical grade) was obtained from Univar, New South Wales, Australia. Apoptosis kits based on staining with annexin V-FITC and propidium iodide (PI) were purchased from Lianke Technology (Hangzhou, China). Emodin, which was used as internal standard was obtained from National Institutes for Food and Drug Control, Beijing, China. 35-mm glass-bottom culture dishes were purchased from NEST Biotechnology Co., Ltd. Jiangsu, China. All other materials and solvents (analytical grade) were obtained from Sigma Aldrich, St Louis, MO, USA.

AGEs were prepared as previously described [21 (link)]. Briefly, bovine serum albumin (BSA; 7 mg/mL) was incubated with glycolaldehyde dimers (90 mM; Sigma-Aldrich, Diegem, Belgium) in sterile PBS (pH 7.4) for 5 days at 37 °C. This solution was dialyzed against PBS, two times for 2 h and overnight at 4 °C to remove unreacted glycolaldehyde (3.4 kDa cut-off). AGEs were filtered (0.2 µm filter, Sarstedt, Antwerp, Belgium). BSA incubated in PBS (7 mg/mL) was used as a control solution.

Cocultures for evolution experiments and subsequent confirmatory coculture experiments were conducted on TSA. After each coculture period each species was isolated on their respective isolation agar, Pseudomonas isolation agar (PIA; BD Difco) and Staphylococcus isolation agar (SIA; TSA BD Difco with 7.5 % NaCl). For liquid cultures, bacteria were cultured in lysogeny broth (LB; Teknova) which was supplemented with erythromycin (25 µg ml⁻¹) to select for transposon mutants and/or with chloramphenicol (10 µg ml⁻¹) to maintain fluorescent plasmids. Chemically defined media with glucose (CDMG) was made according to Hussain et al. [25 (link)], with varying levels of aspartate (1.1 mM or 2.2 mM) and glutamate (1.0 mM or 2.0 mM), as needed (Supplementary Methods, available in the online version of this article). CDMG was always stored at room temperature, in the dark and used within 5 days. Depleted TSB medium, used for single-cell microscopy, was prepared by diluting an overnight culture of PAO1 1 : 100 into 10 ml TSB and growing the culture for either three or 16 h. Cultures were filter sterilized (0.2 µm filter, Sarstedt) to remove cells from the supernatant and referred to in this study as ‘3hr-depleted TSB’ or ‘16hr-depleted TSB’, respectively.

Extracellular vesicles (EVs) from cells were isolated by ultracentrifugation. Cells were grown in EVs-depleted media for 48 h before CM was collected for EVs purification. To isolate total EVs, CM was centrifuged at 300 × g for 10 min to remove cells. EVs were collected by ultracentrifugation at 120,000 × g for 70 min. Apoptotic bodies (ABs), microvesicles (MVs), and exosomes were isolated by differential centrifugation as published before^{84 ,85} . Briefly, the harvested CM was centrifuged at 300 × g for 10 min to remove cells. ABs were collected by centrifugation at 2000 × g for 20 min. The supernatant was centrifuged at 16,500 × g for 20 min to collect MVs. After that, the Supernatant was passed through a 0.2 µm filter (Sarstedt, Nümbrecht, GERMANY) to remove the particles larger than 200 nm. Exosomes were then pelleted by ultracentrifugation at 120,000 × g for 70 min. The isolated EVs were analyzed and quantified by nanoparticle tracking analysis (NTA) and electron microscope with negative staining.