Viability assay

For cell death measurement, cells were seeded in 12-well plates at a concentration in a range of 20,000 to 150,000 cells according to the cell line, so that at the moment of vitamin C treatment, cells reach 40% of confluence. 24 h after seeding, cells were rinsed twice in PBS and then grown in CTR or STS medium. After 24 h, media were refreshed to ensure that glucose and serum levels were not completely exhausted, and after medium pH stabilization at 37 °C and 5% CO₂ atmosphere, cells were treated with 350 μM vitamin C or vehicle for the next 24 h. For experiments with anti-oxidant agents, cells were treated with 5 mM glutathione, and 5 mM N-acetyl cysteine, together with vitamin C. For experiments with DFO, 500 μM DFO was added in CTR and STS media 6 h before vitamin C treatment for HCT116 cells or 12 h before DLD1 and CT26. After the specific incubation time, cells were washed twice in PBS to evaluate only the intracellular effect of DFO in chelating iron and to avoid possible interactions with chemical components in the growth medium, and then, CTR or STS fresh medium and vitamin C were added for the next 24 h for HCT116 and DLD1 cells, and 9 h for CT26. For hydrogen peroxide scavenging experiments, cells were treated with catalase (CAT) from bovine liver (50 U/mL) 1 min before vitamin C was provided as previously described³ or with MnTMPyP (50 µM), 2 h before vitamin C administration.

For HO-1 activation experiments, cells were treated with hemin at a concentration of 20 μM, 3 h before vitamin C was provided. For HO-1 inhibition experiments, cells were treated with zinc protoporphyrin (ZnPP) at a concentration of 20 μM, 3 h before vitamin C was provided. For medium dissection experiments, cells were grown in the following media for a total of 48 h:

-Low concentration of glucose (0.5 g/L) and standard concentration of serum (10% FBS);

-low concentration of serum (1% FBS) and standard concentration of glucose (1 g/L);

-low concentration of glucose (0.5 g/L) and 10% dialyzed FBS;

-STS medium supplemented with IGF-1 (PeproTech, Cat. #: 100-11, 250 ng/ml) or EGF (Biomol, Cat. #: BPS-90201-3, 200 ng/mL) or insulin (Sigma-Aldrich, Cat. #: 11376497001, 200 ng/mL) or their combination with or without glucose (1 g/L);

-STS supplemented with essential amino acids, supplemented as 2× concentration compared with standard medium (Life Technologies, Cat. #: 11130) or non-essential amino acids (Life Technologies, Cat. #: 11140050, 1 mM);

-STS medium supplemented with apo-transferrin (Sigma Aldrich, Cat. #: T2252, 0.3 mg/ml) or holo-transferrin (Sigma Aldrich, Cat. #: T0665, 0.3 mg/mL).

After 24 h media were refreshed and, after medium pH stabilization at 37 °C and 5% CO₂, cells were treated with vitamin C (350 µM). At the end of the experiment cells were harvested by trypsinization, centrifuged and resuspended for a final concentration of 1 × 10⁶ cells per mL. Cell viability was measured by Muse viability assay kit or Erythrosine B exclusion assay.

For Muse viability assay, cell suspension and Muse viability reagent (Merck Millipore, Cat. #: MCH100102) are mixed in 1:10 ratio and, after 5 min of incubation in the dark, viability was analysed by Muse cell analyser. Data are expressed as percentage of dead cells.

For erythrosine B exclusion assay, cell suspension was diluted 1:1 with erythrosin B 0.1% in PBS (Sigma-Aldrich, Cat. #: 200964), then cells were counted in a Bürker chamber, and percentage of cell death was calculated as the number of Erythrosin B-positive cells with respect to the total number of cells.