Fig. 5

Knockdown of CCT4 caused (HR + P)-EMVs to show enhanced oxidative stress and apoptosis in hypoxia-reoxygenated cardiomyocytes. (A) IntaRNA predicted base sites of lncCCT4-2 binding to CCT4. HR-injured AC16 cells were co-cultured with HR-EMVs, (HR + D)-EMVs, (HR + P)-EMVs and sh-lncCCT4-2-(HR + P)-EMVs, respectively, for 24 h: (B) RT-qPCR analysis for CCT4 mRNA expression in AC16 cells. (C) Western blotting analysis of CCT4 protein expression in AC16 cells. (D) Quantitative analysis of CCT4 protein expression. (E) Overexpression of lncCCT4-2 in AC16 cells, actinomycin D and RT-qPCR to detect the stability of CCT4 mRNA. Knockdown of CCT4 expression in AC16 cells and detection of the effect of (HR + P)-EMVs on HR-injured AC16 cells: (F) CCK-8 assay for AC16 cell viability; (G) LDH assay for cell supernatant lactate dehydrogenase activity; (H) Flow cytometry detection of reactive oxygen species (ROS) levels in AC16 cells after DCFH-DA staining. (I) Quantitative analysis of ROS. (J) Flow cytometry detection of apoptotic rate in AC16 cells after Annexin V-FITC/PI double staining. (K) Quantitative analysis of apoptotic cells. (L-M) Western blotting analysis of Bax, Bcl-2, cleaved caspase-3 in AC16 cells, and the relative gray scales calculated by ImageJ software. These data were representative results (n = 3) of three repetitions. *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant. Data are expressed as mean ± SEM.