Ckk 8

To further assess the growth potential of KDM2B in CRC cells, cell proliferation and colony formation assays were employed. For the proliferation assay, 24 h after transfection, HT-29 and DLD-1 cells were seeded at a density of 3 × 10³ cells per well in 96-well microplates in triplicates, and proliferative cells were stained with CKK-8 (Sigma-Aldrich, USA) and measured at 0, 24, 48, and 72 h. Two hours after adding 10 μl of CCK-8 reagent, the absorbance was measured at 450 nm using the Multiskan Go spectrometer (Thermo Fisher, USA). For the cell colony formation assay, 24 h post-transfection the cells mentioned above were harvested and seeded in 6-well plates at a density of 500 cells per well and incubated for 6 days for the formation of colonies. The colonies were fixed and stained with 1% crystal violet for 20 min and then counted.

Initially, 5 × 10³ HCE-F/well were seeded in 96-well plates and left to adhere, overnight, in complete culture medium. In a first set of experiments the following treatments were carried out in SFM: atropine (1, 1.7, and 3.4 mM), for 24, 48, and 72 h. In a second set of experiments, HCE-F cells were treated with 0.15% HA (HMW: 1.8 MDa; Fidia pharmaceutical SpA, Abano Terme, Italy), 0.5% colostrum (NZpurehealth, Auckland, New Zeland), 0.5% 2-Fucosyl-Lactose (Carbosynth, Berkshire, UK, cat. No. OF06739) and two mixes (Mix A: 0.15% HA + 0.5% colostrum; Mix B: 0.15% HA + 0.5% 2FL) in the presence of atropine 1 mM; the medium with the supplements was changed every day for 4 days. Cytotoxicity was measured by the MTT method with the cell counting kit-8 assay (CKK8, Sigma-Aldrich, St. Louis, Missouri, USA, Cat. No. 96992) according to the manufacturer’s protocol.