2 7 dichlorodihydrofluorescein diacetate dcf da

17-DMAG was obtained from Selleckem (Farmingdale, NY, USA); 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA) was purchased from Thermo Fisher Scientific (San Jose, CA, USA); N-acetyl-L-cysteine (NAC) was obtained from Sigma Aldrich (St Louis, MO, USA); SYBR Green was obtained from Promega (Madison, WI, USA); and, Hsp90 siRNA was obtained from Santa Cruz Biotechnology (Dallas, TX, USA).

2′,7′-Dichlorodihydrofluorescein diacetate (DCFDA) (Thermo, MA, United States) and dihydroethidium (DHE) (Invitrogen, MA, United States) were used to detect the levels of intracellular oxidative free radicals and superoxide anions, respectively. Cryostat sections of the heart were incubated with 2 μM DHE in the dark at 37°C for 20 min. H9c2 cells were stained with DCFDA (10 μM) in a dark environment at 37°C for 1 h. Oxidative stress was examined and observed on a fluorescence microscope. DHE fluorescent intensity was further quantified by ImageJ (NIH, MD, United States), and the relative expression was expressed as fold change relative to the control.

Sorafenib was purchased from Cayman Chemical (Ann Arbor, MI). NAC and Z-VAD-fmk were purchased from Sigma-Aldrich (St. Louis, MO). MitoSOX was obtained from Thermo Fisher Scientific (Waltham, MA) and 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA) was purchased from Thermo Fisher Scientific (San Jose, CA).

K562 cells and erythroid progenitors were fixed with 4% paraformaldehyde, permeabilized and stained with fluorescein isothiocyanate (FITC) anti-HbF antibody (AB-19365, Abcam, Cambridge MA) and FITC-isotype IgG antibody (AB-37406). Flow cytometry was conducted on the FACSCanto (BD Biosciences, San Jose CA) as previously published [15 (link)]. To quantify ROS levels, cells were incubated with 2’,7’-dichlorodihydrofluorescein diacetate (DCF-DA) (10μM; Invitrogen, Carlsbad CA) 4 hours before harvest. To determine the effect of SAL on maturation, sickle erythroid progenitors (5x10⁵) were harvested after drug inductions, fixed with 4% paraformaldehyde and then permeabilized in ice-cold acetone/methanol (4:1). The cells were stained with human FITC conjugated transferrin receptor (CD71; ThermoFisher: 11-0719-42) and human FITC conjugated glycophorin A (CD235a; ThermoFisher: 11-9886-42) antibodies and analyzed by flow cytometry.

Baicalein and N-acetyl-1-cysteine (NAC) were purchased from Sigma Chemical Co. (St. Louis, MI, USA). Hydrogen peroxide 30% was purchased from Junsel Chemical Co. (Tokyo, Japan). 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA) was purchased from Invitrogen™ (Waltham, MA, USA). Antibodies against collagen type 1 alpha 1 (Col1A1) (ab34710), matrix metalloproteinase-1 (MMP-1) (ab38929), zonula occludens-1 (ZO-1) (ab96587), occludin (ab216327), and Nrf2(ab31163) were purchased from Abcam Inc (Cambridge, UK). PGC-1α (sc-518025), PINK1 (sc-517353), NQO-1 (sc-32793), claudin-4 (sc-376643), and β-actin (sc-47778) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).