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. 2019 Oct 11;8(10):1241. doi: 10.3390/cells8101241

Figure 3.

Figure 3

PLIN5 regulated cellular reactive oxygen species (ROS) levels. (A,B) Western blot validation of PLIN5 overexpression (A) and RNAi (B) efficiency. (C) HepG2 cells were transfected with PLIN5 siRNAs or negative control siRNAs or PLIN5 expression vector or pcDNA3.1 (control) vector, respectively. Then, the cellular ROS levels were detected by DCFH-DA probes through a microplate reader (Ex = 488 nm, Em = 525 nm). (D) The cells were transfected with PLIN5 siRNAs or negative control siRNAs or PLIN5 expression vector or pcDNA3.1 (control) vector, respectively, and then treated with 200 μM H2O2. Then, the cellular ROS levels were detected by DCFH-DA probes through a microplate reader (Ex = 488 nm, Em = 525 nm). (E) HepG2 cells were transfected with PLIN5 siRNAs or negative control siRNAs or PLIN5 expression vector or pcDNA3.1 (control) vector, respectively. Then, the cellular ROS levels were detected by DHE through a microplate reader (Ex = 535 nm, Em = 610 nm). (F) The cells were transfected with PLIN5 siRNAs or negative control siRNAs or PLIN5 expression vector or pcDNA3.1 (control) vector, respectively, and then treated with 200 μM H2O2. Then, the cellular ROS levels were detected by DCFH-DA probes through a microplate reader (Ex = 488 nm, Em = 525 nm). (G) The cells were treated with 200 μM H2O2. The cytoplasm and mitochondria were isolated respectively, and then cytosolic and mitochondrial cytochrome c levels were detected respectively through Western Blot. (H) The cells were transfected with PLIN5 expression vector or pcDNA3.1 (control) vector, and then treated with 200 μM H2O2. The cytoplasm and mitochondria were isolated, and then cytosolic and mitochondrial cytochrome c levels were detected respectively through Western Blot. GAPDH was the reference protein of cytosolic component and the Porin/VDAC1 was the reference protein of mitochondrial component. (I) The mitochondrial membrane potential (MMP) was detected by JC-1 probes using the flow cytometry method. These experiments were performed in triplicate. * p < 0.05. Ex, excitation wavelength; Em, emission wavelength.