Iwr 1

The iPSCs were differentiated into cardiomyocytes using a 2D monolayer differentiation protocol. Briefly, ~10⁵ undifferentiated cells were dissociated and re-plated into matrigel-coated 6-well plates. Cells were cultured and expanded to 85% cell confluence and then treated for 2 days with 6 μM CHIR99021 (Axon Medchem) in RPMI and B-27 supplement minus insulin (RPMI+B27-Insulin) (Gibco) to activate the Wnt signaling pathway. On day 2, cells were placed in RPMI+B27-Insulin with CHIR99021 removal. On days 3-4, cells were treated with 5 μM IWR-1 (Merck) to inhibit the Wnt signaling pathway. Stocks of retinoic acid (RA) were prepared as 2 mM in DMSO. On day 5-6, differentiated cells were removed from the IWR-1 treatment and placed in RPMI+B27-Insulin+2 μM RA to atrial myocytes. From day 7 onwards, cells were placed and cultured in RPMI and B-27 supplement with insulin (RPMI+B27+Insulin) (Gibco) until beating was observed. Cells were glucose-starved for 3 days with RPMI+B27+Insulin for purification. The iPSC-derived atrial cardiomyocytes (iPSC-aCMs) of day 30-40 after cardiac differentiation were utilized for downstream investigations.

H9 hESCs, purchased from the Institute of Biochemistry and Cell Biology at Shanghai, Chinese Academy of Sciences, were seeded onto 1% matrigel-coated 6-well plates in mTeSR1 medium (STEMCELL Technologies, cat. no. 05850) to 80–90% confluence. H9 hESCs were cultured and differentiated into CMs by using a monolayer-based directed differentiation protocol as previously reported [13 (link)]. Briefly, sequential treatment of Gsk3 inhibitor and Wnt signaling inhibitor was performed to stimulate cardiogenesis. H9 were cultured in mTeSR1 for 4 days before exposure to CHIR99021 (Selleck, cat. no. S1263) on day 0 and IWR-1 (Sigma, cat. no. I0161) on day 3 in RPMI/B27 without insulin medium (Life Technologies, cat. no. A1895601). The culture media were replaced with RPMI/B27 medium from day 7 to day 20. Differentiation was determined by microscopical inspection of cells starting at day 8 of differentiation. Cardiac mesoderm cells could spontaneously develop into functional contracting CMs.

Human embryonic stem cells (hESCs), line H7, were cultured in serum-free mTeSR1 medium (Stemcell Technologies, #85852) on Matrigel-coated culture plates and passaged when cells reached suitable confluence.
Cardiomyocyte differentiation was carried out when cells reached 85–95% confluence. For differentiation, cells were cultured with RPMI/B-27 medium. Gsk3 inhibitor CHIR-99021(12 mM, Selleck, S2924) or a WNT inhibitor IWR-1 (5 mM, Sigma, I0161) was added to the cells on day 0 and 2–3, respectively. On day 4–6, cells were treated with fresh RPMI/B-27 medium. Contracting cells appeared between 7 and 9 days post differentiation and the hESC-derived cardiomyocytes after ≥ 25 days differentiation were used in the experiments. The successful differentiation of myocardial cells can be determined by the appearance of beating cells, as the beating cells and three-dimensional cell bulge can be observed in the plates when the induced differentiation goes well.

The primary mouse hepatic cells were obtained from the 10 weeks old WT or parkin KO mouse liver as described previously [27 (link)]. Briefly, anesthetize the mice by injecting a mixture of Ketamine (80 mg/kg) and Xylazine (5 mg/kg) in 200 μL of saline intraperitoneally. After perfusion with Hank’s Balanced Salt Solution (HBSS)-EGTA solution (0.5 mM EGTA in HBSS, Gibco, Grand Island, NY, USA) pH = 8), collect the liver then cut it to release hepatocytes. The resulting cells were gently pressed through a 100 μm cell strainer (BD, Franklin Lakes, New Jersey). The filtered cells were washed by Dulbecco’s modified Eagle (DMEM) medium and plated into 100 mm² dishes. The cells were grown at 37 °C in 5% CO2-humidified air in DMEM medium that contained 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 mg/ml streptomycin. DMEM, penicillin, streptomycin, and FBS were purchased from Gibco Life Technologies (Grand Island, NY, USA). To confirm the role of parkin deficiency in ethanol-treated mouse primary hepatic cells, the cells from the liver of WT or parkin KO mouse were treated with ethanol (100 mM) for 24 h. To examine whether β-catenin signaling associated with hepatic lipid accumulation, cells were pre-treated with β-catenin signaling inactivator, IWR-1 (10 μM, sigma, St. Louis, MO) for 3 h, then treated with ethanol (100 mM) for 24 h.