Nb100 74340

Human hepatocellular carcinoma (HepG2) cells (American Type Culture Collection, Manassas, VA) were maintained in Advanced Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (Bio International, Auckland, New Zealand), 2 mM L-glutamine, 0.25 μg/mL amphotericin B, 100 U/mL penicillin, and 100 μg/mL streptomycin (Life Technologies) at 37°C in a humidified environment with 5% CO₂. HepG2 cells were seeded at 5×10⁵ cells/mL. 24 hours after seeding, cells were treated with 5 μg/ml of either purified Lp(a) or LDL for 6 hours at 37°C. Cell lysates were harvested and 40 μg of cell lysate protein was subject to western blot analysis with anti-Gpx1 (ab108427, Abcam), anti-Prdx6 (ab16947 Abcam), and anti-SOD (ab13498 Abcam) antibodies. An anti-actin antibody (Novus NB100-74340) was used as a loading control. Protein levels were quantified by densitometry using Image Studio Lite (LI-COR Biosciences, Inc) after normalisation to actin.

FOXO3 western blots were performed as described previously [44 (link)]. Cells were lysed in Triton lysis buffer (50mM Tris-HCL pH 7.5, 100mM NaCl, 0.5 mM EDTA, 0.4% Triton, 50 mM NaF, 40 mM b-glycerophosphate, 1mM sodium orthovanadate, 1mM PMSF, 0.055 units/ml aprotinin). For LC3 western blots, cells were lysed in 150 mM NaCl, 1.0% Triton, 0.5% Sodium Deoxycholate, 0.05% SDS, 50mM Tris-HCL pH 8 and a cOmplete Mini Protease Inhibitor Cocktail Tablet (Sigma) by shaking at 4°C for 30 minutes. Lysates were centrifuged at max speed for 7 minutes (15 minutes for LC3) and supernatants transferred to new tubes. Protein concentration was quantified on a Qbit using the Qbit Protein Assay Kit (Life Technologies), followed by the addition of Laemmli sample buffer (2% SDS, 10% glycerol, 5% β-meracaptoethanol, 63 mM Tris-HCl pH 6.8, bromophenol blue). All antibodies were diluted in 5% powdered milk or BSA and primary antibody incubations were performed overnight at 4°C. Primary antibodies and concentrations used: Rabbit anti-LC3B 1:1000 (Novus Biologicals NB600-1384), Rabbit anti-FOXO3 1:1000 (CST 75D8), Rabbit anti-FOXO1 1:1000 (Abcam ab12161), Rabbit anti-pS6 1:1000 (CST 4858S), Mouse anti-S6 1:1000 (CST 2317S), Rabbit anti-Pink1 1:1000 (Novus BC100-494) Mouse anti-Actin 1:5000 (Novus Biologicals (NB100-74340).

Rabbit-Ca_v1.2 (ACC-003; Alomone Labs), rabbit-RyR2 (MA3-916; Thermo Fisher Scientific), mouse-RyR2 (ab2827; Abcam), mouse-SERCA (NB300-581; Novus Biological), rabbit-ser16-PLN (A010-12; Badrilla), rabbit-thr16-PLN (A010-13; Badrilla), mouse-PLN (A010-14; Badrilla), rabbit-HIF1α (NB100-479; Novus Biological), mouse-HIF2α (clone-190b, MilliporeSigma), rabbit-H3K36me3 (ab9050; Abcam), rabbit-H3K9me3 (ab8898; Abcam), rabbit-H3K4me3 (ab8580; Abcam), rabbit-H3 (ab1791; Abcam), mouse-actin (NB100-74340; Novus Biological), goat anti–rabbit-HRP (sc-2030; Santa Cruz Biotechnology Inc.), goat anti–mouse-HRP (sc-2031; Santa Cruz Biotechnology Inc.), goat anti–rabbit-AlexaFluor 488 (ab15007; Abcam), and goat anti–mouse AlexaFluor 594 (11032; Invitrogen).