Fig 2: Western blot analysis of Cytosolic and mitochondrial fractions of HCC1937 and MCF7 cancer cells. Left panel: (a) HSP60 resulted upregulated in the whole extract of hereditary breast cancer cells (HCC1937) compared to sporadic breast cancer cells (MCF7); Western blot signals were normalized using a-tubulin as housekeeping proteins. (b) HSP60 was down-expressed in the cytosolic fraction of hereditary cancer cells (HCC1937) compared to sporadic breast cancer cells (MCF7); Western blot signals were normalized using a-tubulin as housekeeping proteins. (c) HSP60 was upregulated in the mitochondrial fraction of hereditary breast cancer cells (HCC1937) compared to sporadic breast cancer cells (MCF7); Western blot signals were normalized using cyt c as housekeeping proteins. Right panel: Densitometry analysis for each analyzed proteins. Analysis was performed using three independent experiments. Data are mean ± SEM (N = 3) p < 0.05. For each western blot 50 µg of proteins were resolved by SDS-PAGE using Any kD™ Mini-PROTEAN precast gels and electrotransferred to a nitrocellulose membrane with a Trans-blot turbo system (Biorad). Images were acquired using the Alliance 2.7 system (UVITEC, Eppendorf, Milan, Italy) and analyzed by excel spreadsheet.

Fig 3: (a) BRCA1 western blot analysis. The expression of BRCA1 was assessed by western blot analysis on MCF7, HCC1937 breast cancer cells and MCF10 normal immortalized breast cells. 80 µg of proteins for each cell line were resolved on 4–20% precast polyacrylamide gels (Biorad), electrotransferred to a nitrocellulose membrane with a Trans-blot turbo system (Biorad) followed by immunoblotting. Rabbit monoclonal antibody against BRCA1 (clone D-20, Santa Cruz) was used at a final concentration of 1 µg/mL. HRP-conjugated ?-Tubulin (clone C-20, Santa Cruz) was used at a final concentration of 1µg/mL to ensure equal amount of protein loading. Images were acquired using the Alliance 2.7 system (UVITEC, Eppendorf, Milan, Italy). (b) Mitochondria isolation. Proteins extracts from mitochondrial (M) and cytosolic (C) fractions were analyzed by western blot analysis. The upper blots are representative of cyt c expression in breast cancer cells (MCF7 and HCC1937) as well as in normal immortalized cells (MCF10). The lower blots show a-tubulin expression in breast cancer cells (MCF7 and HCC1937) as well as in normal MCF10 cells. 50 µg of proteins were resolved by SDS-PAGE using Any kD™ Mini-PROTEAN precast gels and electrotransferred to a nitrocellulose membrane with a Trans-blot turbo system (Biorad). Images were acquired using the Alliance 2.7 system (UVITEC, Eppendorf, Milan, Italy).

Fig 4: Analysis of Sarkosyl-insoluble tau from the hindbrain by PHF1 immunoblot.Quantification of immunoblot signal of human and mouse tau doublet at (A) ~50 kDa and (B) ~25 kDa fragment. (C) The ratio of ~25 kDa fragment to ~50 kDa bands. Panel D is a representative blot showing Baseline (lanes 1–4); Vehicle (lanes 5–8), 30 mg/kg dose group (lanes 9–11), and 40 mg/kg dose group (lanes 12–15). The arrow in panel D indicates the 25 kDa tau fragment containing the ptau 396/404 epitope. The same preparations that were evaluated by ELISA in Fig 1 were resolved by 4–20% SDS-PAGE (Criterion TGX Gel, Bio-Rad Laboratories, Inc., Hercules, CA) run with reducing agent and transferred to a PVDF membrane (Trans-Blot Turbo Transfer System, Bio-Rad). The FluorChem R System (ProteinSimple, San Jose, CA) was used to capture images, and quantification of chemiluminescent signal was performed using AlphaView software (ProteinSimple). 30, 30 mg/kg dose group; 40, 40 mg/kg dose group; BL, Baseline; Veh, Vehicle.

Fig 5: Advanced glycation end-products (AGEs) detection on histone from breast cancer cells; (A) Glycolytic activity was measured by the extracellular acidification rate (ECAR). The study was done using the XFp Analyzer. The analysis reveals increased glycolytic activity in HCC1937 breast cancer cells as compared to MCF7 breast cancer cells. The complete report analysis was provided as Supplementary File S1; (B) Histones proteins were extracted in acidic conditions and analyzed using Triton-Acid-Urea (TAU) gel. The method resolves very basic proteins combining two perpendicular separation methods. In the first-dimension histones migrate in function of their isoelectric point an hydrophobic properties, in the second dimension histones are resolved based on molecular weight. Histone spots were excised, trypsin digested and identified by LC-MS/MS analysis. Mass spectrometry analysis was done focusing on methylglyoxal (MGO) as post-translational modifications (PTM); (C) Mass spectrometry analysis of MGO on histones sequences. In the Figure, the main glycated residue on histone sequences is reported. Each identified glycated residue is bold red. MG-H is acronym of methylglyoxal-derived hydroimidazolone. The number under the red residue indicates the position of the amino acid. LC-MS/MS identification data and MS/MS spectra were provided as Supplementary Files S2 and S3; (D) Western blot analysis of MGO-modified histones. Histones proteins were resolved on 15% SDS-PAGE and transferred to a nitrocellulose membrane using the Trans-blot turbo system (Bio-Rad). Membranes were hybridized with primary antibodies against MGO-adducts. Western blot patterns were analyzed using Image Master 2D Platinum software. The analysis allows us to conclude that MGO adducts were more abundant in HCC1937 cells compared to MCF7 breast cancer cells; (E) MGO Western blot signal was normalized against the whole levels of Histone H3.