DDX3X alleviates doxorubicin‐induced cardiotoxicity by regulating Wnt/β‐catenin signaling pathway in an in vitro model

Figure 1. DDX3X expression in Dox‐induced cardiotoxicity in H9c2 cells. (A) The protein level of Casp‐3 and cleaved Casp‐3 in H9c2 cells treated with Dox (2 μM) for 0, 6, 12, and 24 h were detected by Western blot. The data are presented as means ± SEM. ns, p ≥ 0.05 refers to not significant; ***p < 0.001. (B) The apoptosis ratio of H9c2 cardiomyocytes was measured by flow cytometric analysis. The data are presented as means ± SEM. *p < 0.05. (C) The mRNA expression of DDX3X in H9C2 cardiomyocytes treated by Dox (2 μM) for 0, 6, 12, and 24 h were detected by RT‐qPCR. The data are presented as means ± SEM. ***p < 0.001. (D) The protein level of DDX3X in H9C2 cardiomyocytes stimulated by Dox (2 μM) for 0, 6, 12, and 24 h. The data are presented as means ± SEM. ns, p ≥ 0.05 refers to not significant; **p < 0.01

Figure 2. Ddx3x knockdown and pharmacological inhibition of DDX3X both exacerbated Dox‐induced cardiomyocyte apoptosis. “+” symbol denotes presence and “−” symbol denotes absence of the corresponding treatment condition. (A) The RT‐qPCR analysis assessed the efficiency of Ddx3x knockdown. Data are presented as means ± SEM. ***p < 0.001. (B) The protein level of DDX3X, Casp‐3 and cleaved Casp‐3 in H9c2 cells treated with Dox (2 μM) following Ddx3x siRNA transfection. Data are presented as means ± SEM. ns, p ≥ 0.05 refers to not significant; *p < 0.05; **p < 0.01; ***p < 0.001. (C) Flow cytometric analysis detected the apoptosis ratio of H9c2 cardiomyocytes treated with Dox (2 μM) following Ddx3x siRNA transfection. The data are presented as means ± SEM. ns, p ≥ 0.05 refers to not significant; **p < 0.01. (D) Mito‐Tracker Red CMXRos and Hoechst 33342 staining were detected respectively mitochondrial morphology and nuclear condensation of H9c2 cardiomyocytes treated with Dox (2 μM) following Ddx3x siRNA transfection. Scale bar = 50 μm. (E) The CCK‐8 assay was used to detect cell viability of H9C2 cells treated with Dox (2 μM) following Ddx3x siRNA transfection. Data are presented as means ±…

Figure 3. Ddx3x overexpression ameliorated Dox‐induced cardiomyocyte apoptosis. “+” symbol denotes presence and “−” symbol denotes absence of the corresponding treatment condition. (A) The RT‐qPCR analysis evaluated the efficiency of Ddx3x plasmids transfection. Data are presented as means ± SEM. **p < 0.01. (B) The protein level of DDX3X, Casp‐3, and cleaved Casp‐3 in H9c2 cells treated with Dox (2 μM) following Ddx3x plasmids transfection. Data are presented as means ± SEM. ns, p ≥ 0.05 refers to not significant; *p < 0.05; **p < 0.01. (C) Flow cytometric analysis detected the apoptosis ratio of H9c2 cardiomyocytes treated with Dox (2 μM) following Ddx3x plasmids transfection. The data are presented as means ± SEM. ns, p ≥ 0.05 refers to not significant; *p < 0.05. (D) Mito‐Tracker Red CMXRos and Hoechst 33342 staining were detected respectively mitochondrial morphology and nuclear condensation of H9c2 cells treated with Dox (2 μM) following Ddx3x plasmids transfection. Scale bar = 50 μm

Figure 4. DDX3X regulated Dox‐induced cardiotoxicity via canonical Wnt/β‐catenin signaling pathway. “+” symbol denotes presence and “–” symbol denotes absence of the corresponding treatment condition. (A) The protein level of β‐catenin in H9c2 cells treated with Dox (2 μM) for 0, 6, 12, and 24 h. Data are presented as means ± SEM. ns, p ≥ 0.05 refers to not significant; *p < 0.05. (B) The protein level of β‐catenin in H9c2 cells treated with Dox (2 μM) following Ddx3x siRNA transfection. Data are presented as means ± SEM. ns, p ≥ 0.05 refers to not significant; *p < 0.05; **p < 0.01. (C) The protein level of β‐catenin in H9c2 cells treated with Dox (2 μM) following RK‐33 pretreatment. Data are presented as means ± SEM. ns, p ≥ 0.05 refers to not significant; *p < 0.05; ***p < 0.001. (D) The protein level of β‐catenin in H9c2 cells treated with Dox (2 μM) following Ddx3x plasmids transfection. Data are presented as means ± SEM. *p < 0.05; **p < 0.01. (E) The protein level of DDX3X, β‐catenin, Casp‐3, and cleaved Casp‐3 in H9c2 cells treated with Dox (2 μM) following Ddx3x siRNA transfection and Licl pretreatment. Data are presented as means ± SEM. ns, p ≥ 0.05 refers to not significant; *p…