Wst 1

Proliferation of HRMEC was determined using a 5-bromo-2’-deoxyuridine (BrdU) ELISA in accordance to the manufactures recommendations (Roche, Mannheim, Germany). In brief, 1.2x10⁴ cells/cm² HRMEC were seeded onto a 96 well plate and incubated for 24 h to achieve complete adherence of the cells. After incubation with hr-GAL1 or 3 and BrdU labeling solution for 48 h, cells were fixed and incubated with anti-BrdU antibodies in accordance to the manufacturer’s instructions. Following the substrate reaction, the product formation was quantified by absorbance measurement at a wavelength of 450 nm and a reference at 690 nm on the SpectraMax 190 ELISA reader (Molecular Devices, San Jose, CA).
To analyze cell viability, a colorimetric dye reduction assay was conducted matching the manufacturer’s recommendations (WST-1; Roche). In brief, 1,5x10⁴ cells/cm² were seeded and incubated for 24 h. After treatment of HRMEC with hr-GAL1 or 3 for 72 h, WST-1 was added and incubated for additional 2 h. For readout, absorbance measurement at a wavelength of 450 nm and a reference at 690 nm was determined on a SpectraMax 190 ELISA reader (Molecular Devices).

HEK-293 cells were treated with afatinib and compounds 10b, L1, and L3 at the indicated doses for 36 h and analyzed using the cell proliferation reagent WST-1 [2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazoliumwater-soluble tetrazolium salt] (Roche Molecular Biochemicals, Basel, Switzerland). Samples’ absorbance of WST-1 was determined using an ELISA reader (Molecular Devices LLC, San Jose, CA, USA) at 450 nm according to the manufacturer’s instructions.

HCC cell growth rates were measured using the WST-1 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1, Sigma-Aldrich, CA, USA) method. First, the cells were seeded in a 96-well plate at 5 x 10³ cells/well, incubated at 37°C for 24 hours, transferred to serum-free medium, and transfected with siRNA as described above. Then, after cultivation under 21% O₂ for 24 hours, the cells were transferred to a culture medium containing 10% FBS. Finally, WST-1 was added to the wells, and the plates were incubated at 37°C for 3~4 hours for efficient cell dyeing, and analyzed for its absorbance at 460 nm using a spectrophotometer (Molecular Devices, USA).

The interaction effect on cell proliferation was measured with WST–1 (Roche). Cells (1 × 10⁴ cells/100 µL) were treated with ZnO NPs suspended in DW, MEM, or each food matrix solution. All food matrices (1 mg/mL) for cell experiments were prepared in MEM by stirring for 30 min. After incubation for 24 h, 10 µL of WST-1 solution was added to each well and further reacted for 4 h. The absorbance was measured at 440 nm versus 650 nm using a microplate reader (SpectraMax M3, Molecular Devices, Sunnyvale, CA, USA).

Cell Growth Assay. Breast cancer cells growth rates were measured using the WST-1 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1, Sigma-Aldrich, CA, USA) method. First, the cells were seeded in a 96-well plate at 5 × 103 cells/well, incubated at 37 °C for 24 h, transferred to serum-free medium, and transfected with S63A and S262A as described above. Then, after cultivation under 21% O2 for 24 h, the cells were transferred to a culture medium containing 10% FBS. Finally, WST-1 was added to the wells, and the plates were incubated at 37 °C for 3 ~ 4 h for efficient cell dyeing, and analyzed for its absorbance at 460 nm using a spectrophotometer (Molecular Devices, USA). And, cell viability was measured by tryphan blue staining. It is based on the principle that live cells possess intact cell membranes that exclude certain dyes, such as trypan blue. When a cell suspension is simply mixed with the dye, add equal parts of 0.4% trypan blue dye to the cell suspension to obtain a 1 to 2 dilution, and incubate mixture for less than 3 min at room temperature. Place the hemacytometer with 1:1 tryphan blue staining cells on the stage of a light microscope (Olympus Optical Co., Tokyo, Japan) and calculated the staining cells.

The treatment effects on metabolic activity of tumor cells were measured using the tetrazolium-salt-based reagent WST-1 (Roche, Basel, Switzerland). Cells were seeded in 24-well plates (3000 cells/1 mL culture medium per well) and treated with the following doses/concentrations: IEPA single or daily (4 fractions), 0.1–100 µM; IR daily, 4 fractions, 4 × 0.5–2.8 Gy (FaDu) or 4 × 1.6–8.2 Gy (A172); CIS, 0.1–30 µM (FaDu only); TMZ daily, 4 fractions, 5–100 µM (A172 only) and/or CCNU, 1–50 µM (A172 only). Seventy-two hours after the last treatment fraction, WST-1 assay was carried out according to manufacturers’ instructions and absorbance was measured at 435 nm with a reference wavelength of 680 nm on a spectrophotometer (SpectraMax i3x, Molecular Devices, LLC, San José, CA, USA) after thirty minutes.
ID₅₀ and IC₅₀ values were determined using exponential regression in Microsoft Excel 16.0 or, if not feasible, estimated directly from the graph of metabolic activity reduction (Table 1, Figure 1). For fractionated IR, experimentally determined ID₅₀ values of both cell lines were averaged.