Lysis solution

The LoVo and SW480 cells or exosomes were collected, lysed with lysis solution (Roche) on ice for 30 min, and the total protein was separated by centrifugation at 14000 rpm for 30 min. Afterward, 50 μg total protein was loaded on 12% polyacrylamide gel and went through 2-hour electrophoresis at 100 V. The separated protein was then transferred to polyvinylidene fluoride (PVDF) membranes. After being blocked with 5% skimmed milk at room temperature (RT) for 1 hour, the membranes were washed with TBST 3 times (10 min each time) and incubated with the antibodies (all from Abcam, MA, USA) of CD9 (ab263019, 1: 1000), CD63 (ab271286, 1: 1000), HSC70 (ab76005, 1: 1000), THEM4 (ab106435, 1:1000), p-AKT (ab38449, 1:1000), AKT (ab8805, 1:500), p-NF-κB (phospho S536) (ab106435, 1: 1000), NF-kB (ab32536, 1: 1000), and GAPDH (ab8245, 1: 1000) at 4°C overnight. After the membranes were rinsed with TBST, they were incubated with horseradish peroxidase (HRP)-labeled anti-rabbit secondary antibody (concentration 1:300) for 1 hour at RT. Next, TBST was employed to wash the membranes 3 times (10 min each). At last, a Western blotting reagent (Invitrogen) was used for blots imaging, and the gray intensity of each protein was determined via Image J.

When the cells reached 80–90% confluence, Hct116 cells, LoVo controls, and PDCs were collected. The cells were lysed on ice with approximately 100 µL of lysis solution (Roche, Germany) for 30 min and then centrifuged at 4 °C and 14,000 rpm for 30 min. After determining the protein concentrations in the samples, the proteins were boiled at 100 °C for 10 min and separated on a 10% sodium dodecyl sulfate polyacrylamide gel. Protein bands were transferred onto polyvinylidene difluoride membranes (GE Healthcare, USA), which were blocked with 5% skim milk (BD Biosciences, USA) at room temperature for 90 min. The membranes were then incubated overnight with the respective primary antibodies (Additional file 1: Table S1) at 4 °C. Subsequently, the membranes were incubated with secondary antibodies at room temperature for approximately 2 h. β-Actin and histone-H3 were used as protein loading controls. Protein expression was detected using a ChemiDoc Imaging System (Bio-Rad, USA).

Transiently transfected NSC-34 cells in 6-well plates (7 × 10⁵ cells/well) were washed twice with DMEM/F12 medium and pre-incubated in the absence or presence of 10 µM AZ10606120 in DMEM/F12 medium for 1 h at 37 °C/5% CO₂. Cells were then incubated in the absence (basal) or presence of 5 mM ATP in DMEM/F12 medium for 20 min at 37 °C. Cells were visualised by differential interference contrast imaging using an Eclipse TE2000 inverted microscope with images captured using Image-Pro AMS 6.1. The media from wells were collected and the remaining adherent cells lysed with Lysis Solution (Roche, Mannheim, Germany). Lactate dehydrogenase (LDH) release and percent cytotoxicity were determined using the Cytotoxicity Detection KitPLUS (Roche) as per the manufacturer’s instructions using a Molecular Devices (San Jose, CA, USA) SpectraMax Plus 384 Microplate Reader. The percentage of LDH release was determined as the amount of LDH released into the medium over the total amount of LDH released in each sample.