Figure 4.

Knockdown of MALAT1 Induces Mitochondrial Apoptosis

(A) Abnormal mitochondrial structure in MALAT1 knockdown HepG2 cells. Scale bar = 0.5 μm. Under TEM, mitochondria had swollen vesicular matrix compartments and small vesicular matrix compartments surrounding a swollen vesicular compartment. (B) Mitochondria apoptosis biomarkers. Western blot was used to measure the biomarkers for mitochondrial apoptosis, including Bcl-2, Bax, Caspase-3, and cytochrome C. β-Actin and MT-CO1 were used as the cytoplasmic and mitochondrial controls. (C) Quantitation of western blot results. Data are presented as mean ± SEM. Significant differences were determined by one-way ANOVA followed by Student’s t test. ∗∗∗∗p < 0.001 compared with the shCT group. (D) FACS analysis of cell death. Standard dot plot diagram from FACS shows the progression of cell death. Q1, double negative (annexin V and 7-AAD negative) represented healthy cells; Q2, annexin V positive and 7-AAD negative represented apoptotic cells; Q3, annexin V &7-AAD double positive represented necrotic cells. (E) Percentage of cell population. Apoptotic, healthy, and necrotic cells were quantitated by flow cytometry. ∗∗∗∗p < 0.0001 compared with the shCT control group. (F) Mitochondria membrane potential. The membrane potential was measured by JC-1 staining. FCCP was used as the positive control of cell apoptosis. In cells with high mitochondrial membrane potential, JC-1 spontaneously forms complexes with intense red fluorescence. In apoptotic or unhealthy cells, JC-1 was stained with green fluorescence. The ratio of green to red fluorescence is associated with the membrane potential. ∗∗∗∗p < 0.0001, ∗∗p < 0.01 compared with the shCT control group.