Brdu incorporation elisa assay

Cell proliferation was analyzed by the BrdU incorporation ELISA assay following the manufacturing instructions (Roche Diagnostics GmbH, Mannheim, Germany). The incorporation of BrdU into genomic DNA during cell growth was quantified and analyzed after SPGE extract treatment (10–1,000 μg/mL) in our experiments. After the substrate degradation, the color intensity changes were read at 450 nm (reference wavelength: 690 nm) by Cytation^TM 3 Cell Imaging Multi-Mode Reader (Biotec). The numbers of experimental repeats were set (n = 3). Based on Resazurine and BrdU assays, the final IC₅₀ was calculated and determined: 160 μg/mL (MCF-7 cells) or 130 μg/mL (MDA-MB-231 cells) (Kubatka et al., 2019 (link); Kubatka et al., 2020a (link); Kubatka et al., 2020b (link)).

In this study was used: anti-rabbit and anti-goat secondary antibodies as well as polyclonal RIP1, RIP3, Bax and Bcl-2 antibody from Santa Cruz Biotechnology (Santa Cruz, CA); polyclonal LC3 antibody from Abcam; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and RNAse A and 2′,7′-Dichlorofluorescin diacetate (DCF-DA) from Sigma-Aldrich; Annexin V-FITC apoptosis detection kit and propidium iodide staining solution from BD Pharmingen; BrdU incorporation ELISA assay from Roche (Grenzach-Wyhlen, Germany).

The reagents used for the syntheses were of analytical grade and were used without further purification. They were as follows: VO(acac)2 (≥98%, Sigma-Aldrich, Saint Louis, MO, USA), N,N-bis(carboxymethyl)-dl-alanine trisodium salt (Na3bcma) (≥90%, Sigma-Aldrich), N-(Phosphonomethyl)iminodiacetic acid (H4pmida) (95%, Sigma-Aldrich), N-(2-carboxyethyl)iminodiacetic acid (H₃ceida) (≥98.0%, VWR) and 4-amino-2-methylquinoline (4-NH2-2-Me(Q), ≥98%, Sigma-Aldrich). In a biological study, it was used anti-rabbit secondary antibodies as well as polyclonal anti-p53 and RAGE antibodies from Santa Cruz Biotechnology (Dallas, TX, USA). Polyclonal anti-cdk1, cyclinB1, p21, β-actin and LC3β antibodies were purchased from Abcam (Cambridge, UK). Anti-rabbit antibodies conjugated with Alexa Fluor546 were obtained from Thermo Fischer Scientific (Waltham, MA, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), neutral red (NR), RNAse A, 2′,7′-dichlorofluorescin diacetate (DCF-DA), phalloidin, hoechst, bovine serum albumin (BSA), paraformaldehyde, gemcitabine, and propidium iodide were purchased from Sigma-Aldrich (Saint Louis, MO, USA). BrdU incorporation ELISA assay from Roche (Grenzach-Wyhlen, Germany) and LDH-Cytotoxicity Assay from Promega (Madison, WI, USA).