Keap1

Cells on glass coverslips were fixed with 4% paraformaldehyde (Sigma) for 20 min at 37 °C, permeabilized with 0.3% Triton X-100 (Sigma, cat#T8532), blocked with 3% bovine serum albumin (BSA; ThermoFisher, cat#15260037), and stained appropriately before finally being mounted with the Antifade mounting medium plus DAPI (Vectashield, Burlingame, CA, USA, cat#H1200). The cells were stained with the following primary antibodies overnight at 4 °C: Nrf2 (Abcam, ab137550) (1:100); Keap1 (ThermoFisher, MA5-17106) (1:100). After several washes in 0.3% triton in PBS, the cells were incubated in the dark for one hour at room temperature with secondary antibody. Secondary antibodies were as follows: Alexa Fluor 488 donkey anti-rabbit IgG and Alexa Fluor 555 donkey anti-mouse IgG (Life Technologies, 1:500). After several washes, cells were mounted before imaging. Cardiac troponin T (cTNT; Invitrogen, cat#MA5-12960) (1:100) was used to stain iPSC-CMs. Images were taken on a Nikon 55i fluorescence microscope.

The reagent contained mangiferin, doxorubicin, dimethyl sulfoxide (DMSO), SDS, tris, glycine, BSA, 3-(4,5-dimethythiazol-2-yl)-2,5-diphenylTrazolium Bromide (MTT), and CelLytic (Sigma-Aldrich, St. Louis, MO, USA). Other ingredients included Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), antimycotic/antibiotic mixtures (Carlsbad, CA, USA), DCFH2-DA, and TUNEL staining kits (Roche, Mannheim, Germany), and antibodies were purchased from Invitrogen, Carlsbad, CA, USA. Antibodies to AKT, pPI3K, PI3K PERK1/2, ERK1/2, pP38, P38, BCL2 PARP, HO-1, NQO1, Keap-1, pNrf2, COX, TNF-α, pNFC-B, β-actin, and Lamin B1 were sourced from Invitrogen and Thermo Fisher, Waltham, MA, USA. The antibody against cleaved caspase-3 was purchased from Abcam (Branford, CT, USA).

Total proteins extracted from the cells was separated by 8–12% SDS-PAGE and transferred onto nitrocellulose membranes, which were incubated with specific antibodies overnight at 4 °C, probed with HRP-conjugated secondary antibodies, and visualized using enhanced chemiluminescence reagent (Pierce). β-actin was used as the loading control. The following antibodies were used: TRIM15 (Cat: PA5-40946, Invitrogen), Keap1 (Cat: PA5-99434, Invitrogen), Nrf2 (Cat: PA5-27882, Invitrogen), anti-6x-His (Cat: MA1-21315, Invitrogen), anti-HA (Cat: 32-6700, Invitrogen), NQO1 (Cat: MA1-16672, Invitrogen), anti-K48-linkage Specific Polyubiquitin (Cat: #8081, Cell Signaling Technology), anti-K63-linkage Specific Polyubiquitin (Cat: #5621, Cell Signaling Technology).