*4.9. Real-Time PCR*

Real-time PCR was performed using the QuantStudio 6 Flex Real-Time PCR System (ABI, Thermo Fisher, Shanghai, China) and Roche LightCycler ® 480 (Roche, Switzerland), and the following PCR program: Denaturation at 95 ◦C for 10 min; amplification for 45 cycles at 95 ◦C for 15 s; annealing and extension at 60 ◦C for 1 min. 2× SYBR Green qPCR Master Mix (#B21203, Bimake, Shanghai, China) were used for the detection of RT-qPCR. Primer sequences are shown in Table 1. Specific amplifications for certain PCR reactions were assessed using a melting curve. One negative control reaction, in which the cDNA template was replaced by water, was performed to avoid potential contamination. The sample from each well was repeated three times, and the comparative Ct (2−ΔΔCt) value method was used for relative quantification. GAPDH (NM\_002046.6) was used as the reference gene.


**Table 1.** Primer used for SYBR Green I qRT-PCR validation.

#### *4.10. Apoptosis and Mitochondrial Membrane Potential Analysis*

The analysis was performed by Servicebio Co., Ltd. (Wuhan, China). Briefly, the cells with transfections were collected through trypsin digestion. Then, the cells were incubated by annexin V-FITC and propidium iodide (PI). Then, the apoptosis rates were detected through flow cytometry (Ex = 488 nm, FL1 (Em = 525 ± 20 nm) and FL2 (Em = 585 ± 21 nm)). Flow Jo software was used to analysis the rates of cells in di fferent conditions. For mitochondrial membrane potential (MMP) analysis, JC-1 probe was used. Briefly, the cells were collected and counted. The cells were then incubated with 10 μg/mL JC-1 probe at 37 ◦C for 20 min. Cells were detected through flow cytometry (Ex = 488 nm, FL1 (Em = 525 ± 20 nm) and FL2 (Em = 585 ± 20 nm)). Flow Jo software was used for the analysis.

#### *4.11. Bioinformatics and Data Analysis*

The survival predication was performed using the GEPIA database (http://gepia.cancerpku.cn/). The prognosis analysis and gene expression analysis were performed according to the construction of the creator of this database [59].

#### *4.12. Dual-Luciferase Reporter Assay*

The promoter region of *PLIN5* was amplified by PCR with total DNA of HepG2 cells. The primers were used as following, F: 5-GAAAACTGGATCGGATGAATTGG-3 and R: 5-CACCCCCGCCGGTCCCGC-3. Then, the promoter region was cloned into the pGL3-basic vector. Then, the reconstructed vector was co-transfected with the ATFs expression or pcDNA3.1 (control) vectors and TK vector into HepG2 cells seeded into the 12-well plate. Moreover, pGL3-basic vector was used as the negative control and pGL3-CMV vector was used as the positive vector. Luciferase enzymatic activity was measured by a microplate reader from a multi-wavelength measurement system (PE Enspire, PerkinElmer, Germany) using a dual-luciferase reporter assay system (#RG027, Beyotime Biotechnology, Nanjing, China). The relative light unit (RLU) was normalized by a control group. The result was showed by the relative RLU. All transfections were performed in triplicate, and the data are expressed as the means ± SD.
