Figure 5.

Increased expression levels of the ROS-producing NOX family enzyme NOX4 and its accessory regulatory subunit p22^phox in Dystrophin-deficient hiPSC-CM cultures

(A) Gene expression profiles at day 24 of cardiac differentiation of NOX2 and NOX4, and the regulatory subunits (p22^phox, p47^phox, p67^phox, RAC1, RAC2, and RAC3) in DMD, DMD isogenic, and healthy control hiPSC-CMs upon treatment with NAC, PTC124, and idebenone. Each data point is represented as ΔCt and is normalized for the housekeeping genes (GAPDH and RPL13a). Data are representative of five or more independent experiments (n ≥ 5), and values are expressed as mean ± SEM. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001 versus subjects within the treatment condition or ^$p < 0.05, ^$$p < 0.01, ^$$$p < 0.001, and ^$$$$p < 0.0001 versus treatment conditions within the subject group.

(B) Representative flow cytometric analyses at day 15 of differentiation showing the percentage of NOX4 (APC) protein expression in SIRPA (PE)-positive DMD hiPSC-CMs upon treatment. Data are representative of three independent experiments (n = 3). Flow cytometry data are reported as mean ± SEM.

(C) Flow cytometric quantification at day 15 of differentiation of the percentage of SIRPA-positive hiPSC-CMs expressing NOX4 upon treatment. Data are representative of three independent experiments (n = 3), and values are expressed as mean ± SEM. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001.

(D) Western blot analysis quantifying the protein expression levels of NOX4 in 15-day-old differentiated DMD and control hiPSC-CMs, normalized to the loading protein ACTB. See also Figures S4I and S4J.