Transwell assay insert

The fetal bovine serum (FBS) was purchased from Gibco (Grand Island, NY, United States). BCA protein assay kit (No. P0011) and One Step TUNEL Apoptosis Assay Kit (No. C1089) were purchased from Beyotime (Shanghai, China). Transwell assay inserts (No. 354483) were purchased from Corning (Corning New York, NY, United States). Enhanced Chemiluminescence (ECL) was purchased from Thermofisher (Waltham, Massachusetts, United States). 1% crystal violet solution (No. V5265) was purchased from Sigma (St. Louis, MO, United States). Anti-rabbit IgG, HRP-linked Antibody, primary antibodies against E-cadherin, Vimentin, p-AMPKα, p-p44/42 MAPK, p-MEK1/2, p-AKT, AKT, p-mTOR, mTOR, p-IκBα, IκBα, p-NF-κB p65, NF-κB p65 and GAPDH were purchased from Cell Signaling Technology (Danvers, MA, United States). Primary antibody against Wnt 3a was purchased from Abcam (Cambridge, United Kingdom). The catalogue numbers of antibodies were shown in Supplementary Table S1. FITC Annexin V Apoptosis Detection Kit (No. 556547) and DNA Reagent Kit (No. 340242) were purchased from BD Biosciences (San Diego, CA, United States). Cell Titer 96^® AQueous One Solution Cell Proliferation Assay (MTS, No. G3581) was purchased from Promega (Madison, WI, United States). The macroporous resin (No. M0032) was purchased from Solarbio Life Science (Beijing, China).

To measure cellular migration, researchers used Transwell assay inserts (Corning, NY, USA). In a 24-well plate, JBMMSCs (8 × 10³ cells in 400 μL serum-free media) were attached to the upper part of the inserts, with 750 μL of media containing various exosomes at a concentration of 50 μg/ml in the bottom chamber. Cells were allowed to culture for 36 h, during which all cells that had migrated to the bottom chamber were fixed with 4% neutral-buffered formalin, followed by staining using crystal violet (0.1%, Solarbio). The cells were then counted and imaged using a microscope.

Transwell assay inserts (Corning) were used as upper chambers to determine the invasive and migratory ability of glioma cells. To detect invasion, the upper chambers were coated with diluted Matrigel (BD) for 12 h at 37 °C. The bottom chambers were filled with 700 μL medium containing 10% FBS as a chemoattractant. Treated glioma cells (transfected, co-cultured or incubated) (1 × 10⁴ cells) were resuspended in 100 μL serum-free medium after serum starvation for 24 h and then seeded into the upper chamber. After incubation for 24 h (migration assay) or 48 h (invasion assay), cells that moved to the lower side of inserts were fixed with 4% paraformaldehyde for 20 min and stained with crystal violet solution for 15 min. Stained cells were imaged and counted with an inverted microscope (Olympus). Cell numbers from five random fields of each group were used for statistical analysis to measure invasion and migration.

About 4×10⁴ Hpa-V cells were seeded in the upper chamber of transwell assay inserts (pore size of 0.4 mm; Corning) with 1mL serum-free DMEM medium, while 5×10⁵ MDA-MB-231 or SkBr3 cells with DMEM medium containing 10% FBS were added into the lower chamber by a 6-well transwell chamber. The co-cultured MDA-MB-231 or SkBr3 cells were named MDA-MB-231/Hpa-V or SkBr3/Hpa-V. After 48 hours, MDA-MB-231/Hpa-V, SkBr3/Hpa-V and MDA-MB-231, SkBr3 cells on the lower chamber were collected to perform the following experiments.

Transwell assay inserts (Corning, NY, USA) were used to assess cellular migration. Briefly, ECFCs (8 × 10³ cells in 400 μL serum-free media) were added to the upper portion of these inserts in a 24-well plate, with 700 μL of complete media in the lower chamber. Cells were cultured for 24 h, after which 4% neutral-buffered formalin was used to fix all cells that had migrated to the lower chamber, which were subsequently stained using 0.1% crystal violet (Solarbio). Cells were then imaged via microscopy and counted.