U87mg

The experiments were performed on normal and cancer cell lines, purchased from ATCC^®, Washington, DC, USA (U87MG, EOC2).
U87MG cells were cultured in DMEM (Sigma-Aldrich, Weinheim, Germany), supplemented with 10% FBS and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin). EOC2 cells were cultured in DMEM, supplemented with 10% FBS. Cells were cultured in a humidified atmosphere at 37 °C, saturated with 5% CO₂.
To remove the adhesive cells from the plates, we used a trypsin/EDTA solution (0.05% of trypsin/EDTA; Sigma-Aldrich, Weinheim, Germany) for U87MG, and a cell scraper for EOC2.
The cells were collected by centrifugation (125× g for 10 min) and placed in a fresh medium without antibiotics prior to treatment with the respective substance. The cells (3 × 10⁵ cells/mL) were incubated with the drug for different time intervals in a cell incubator. At each time interval, aliquots were used for analyses.
Ascorbate was dissolved in PBS (10 mM, pH 7.4). Menadione was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, Weinheim, Germany) to 10 mM stock solutions, and then several working solutions in PBS were prepared. The final concentration of DMSO in the cell suspension was below 1%. At this concentration, DMSO did not affect cell viability.

The human tumor cell lines SW480 (primary colon adenocarcinoma), SW620 (metastatic colon adenocarcinoma), MDA-MB-231 (metastatic breast adenocarcinoma) and U87-MG (glioblastoma) were purchased from Sigma-Aldrich. Cells were grown in two different Dulbecco’s modified essential media (DMEM high glucose for SW480 and SW620 and DMEM low glucose for MDA-MB-231 and U87-MG) (Sigma-Aldrich) supplemented with 10% FBS (Gibco) and 1% penicillin/streptomycin solution (Gibco) at 37 °C with 5% CO₂ in humidified air.
To activate TLR4 cells were incubated with [1 µg/ml] LPS (E. coli 055:B5 LPS, Sigma-Aldrich) for 24 hours, washed three times with PBS and then culture medium was replaced with fresh medium supplemented with 10% certified exosomes-free serum (Gibco). After 24 hours, supernatants were collected and stored at −20 °C until use, while cells were harvested, and their proliferation/viability was determined by the trypan blue exclusion assay.
Cell homogenates were analyzed for TLR4 expression by immunoblotting and the human anti-TLR4 (1:500, Cell Signaling) and anti-β-actin (1:10,000, Sigma-Aldrich) were used as primary antibodies.
All methods of analysis were carried out in accordance with the relevant guidelines and regulation with appropriate quality control.