Ultra low attachment 6 well dishes

The human lung cancer A549 cells were obtained from Cell Bank of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China) and maintained in RPMI 1640 medium supplemented with 10% heat-inactivated foetal bovine serum. All the cells were incubated in a humidified atmosphere with 5% CO₂ at 37°C.
A549 cells were seeded in ultra-low attachment 6-well dishes (Corning) and maintained in serum-free DMEM/F12 (Hyclone Beijing, China) medium supplemented with 20 ng/mL EGF, bFGF and IGF1 (PeproTech, Rocky Hill, NJ, USA). After adding growth factors (20 ng/ml) for two other days, the propagated spheroid bodies were collected for subsequent experiments. At the same time, A549 cells were cultured in ordinary 6-well dishes with the above medium and growth factor as control.
Adenoviruses ZD55-EGFP and ZD55-TRAIL were previously constructed 31 . Embelin and Resveratrol were obtained from Sigma-Aldrich. LY294002 was purchased from Cell Signaling Technology (Beverly, MA, USA).

Spheroid formation assays were performed to evaluate the regulation of cancer stem-like cells (CSCs). OSCC cells were dissociated and suspended in spheroid medium consisting of serum-free DMEM/F12 (Gibco, USA), human bFGF (20 ng/ml, Sino Biological Inc., China), human EGF (20 ng/ml, Sino Biological Inc., China), and B-27 supplement (Life Technologies, USA) in 6-well ultra-low attachment dishes (Corning, USA) under different treatment for 10 days [15 (link)]. The spheroid morphology was observed microscopically, and the number of spheroids was counted and compared.

Single cells were resuspended in serum-free DMEM/F12 (Welgene) containing 1 × B27 supplement (Invitrogen, Waltham, MA), 20 ng/mL epidermal growth factor, and 10 ng/mL basic fibroblast growth factor (PeproTech, Rocky Hill, NJ). Primary spheres were derived by plating 50,000 to 100,000 single cells per well into 6-well ultra-low attachment dishes (Corning, Corning, NY). Dishes were cultivated for 7 days prior to in vivo cell injection.

Total mRNA was reverse‐transcribed into cDNA (AT301 TransGen Biotech, Beijing, China), and real‐time quantitative PCR was performed with CFX96 Real‐Time PCR Detection System (Bio‐Rid, Hercules, CA, USA). For western blot, the protein from cell extracts was separated by 10% SDS‐PAGE electrophoresis and was later transferred onto PVDF membrane. Membranes were incubated with ESR1 (1:3000, EPR4097, ab108398; Santa Cruz, Dallas, TX, USA), TCF4 (1:2000, ab217668; Abcam, Cambridge, MA, USA), CMYC (1:2000, ab32072; Abcam), LEF1 (1:2000, ab137872; Abcam), WNT1 (1:1000, ab15251; Abcam), CTNNB1 (1:1000, C2206; Sigma‐Aldrich, St. Louis, MO, USA), VINCULIN (1:5000, #18799; Cell Signaling Technology, Danvers, MA, USA), and then detected using ECL Blotting Detection Reagents (Merck Millipore, Burlington, MA, USA). Cells of different groups were suspended in DMEM/F12 Medium supplemented with 20 ng/mL EGF (BD Biosciences, San Jose, CA, USA), bFGF and 4 μg/mL insulin (Sigma), and then plated in 6‐well ultra‐low attachment dishes (1000 cells/mL; Corning Incorporated, Tewksbury, MA, USA). To analyse the self‐renewal ability, sphere number of each captured image was counted by using phase contrast microscope (Nikon, Tokyo, Japan).

Macrophages were seeded in 6-well ultra-low attachment dishes (Corning) at 5 × 10⁶ cells/well, followed by the addition of sEVs (final concentration 100 ng/ml) or recombinant proteins. Recombinant human annexin A1 (Abcam, Cambridge, UK; final concentration 100 ng/ml), recombinant galectin-3 binding protein (LG3BP) (Aviva Systems Biology, San Diego, CA, USA; final concentration 1 ng/ml), recombinant human lactoferrin (lactotransferrin) (Abcam; final concentration 0.1 ng/ml), recombinant human lactadherin (MFGE8) (R & D Systems, Inc.; final concentration 100 ng/ml), and recombinant human aminopeptidase N/CD13 (R & D Systems; final concentration 100 ng/ml) were used. After 48 h, macrophages were harvested and mRNA expression levels of genes encoding pro- (Il-6, Tnf-a, Mcp-1, Inos) and anti- (Il-10, Ym-1, Fizz-1, Cd206) inflammatory factors were evaluated using real-time PCR.