*4.4. Transfection Assay*

Cells were seeded on a 6-well plate or on slides in a 24-well plate. Then, the cells were transfected with Lipo8000 ™ Transfection Reagent (#C0533, Beyotime, Nanjing, China). For the preparation of RNAi working solution, 10 μL siRNA oligo (20 μM, Ribobio, Guangzhou, China) was mixed with 10 μL Lipo8000 regen<sup>t</sup> in 100 μL DMEM. For the preparation of the overexpression working solution, 2.5 μg plasmid was mixed with 4 μL Lipo8000 regen<sup>t</sup> in 50 μL DMEM. The working solution was added in the plate well and incubated for 6 h. Then, the plate well was changed with fresh cultural medium (DMEM with 10% FBS) for another 48 h of culture.

#### *4.5. Plasmid DNA Construction*

For the overexpression assay and the localization assay, expression vector and fluorescence-labeled vector were constructed. In brief, the *PLIN5*/*ATF1*/*ATF4* CDS region was amplified by the cDNA library of HepG2 cells using KOD-Plus-Neo DNA polymerase (#KOD-401, TOYOBO, Shanghai, China). After gel extraction, the *PLIN5* CDS fragment was cloned into the digested pcDNA3.1 vector (digestion sites, HindIII and BamHI) using a seamless cloning kit (#C112-01, ClonExpress II One Step Cloning Kit, Vazyme, Nanjing, China). For the localization assay, the gene CDS region was cloned into the digested pCMV-C-Dsred (#D2624, Beyotime Biotechnology, Nanjing, China) or pCMV-C-EGFP (#D2626, Beyotime Biotechnology, Nanjing, China). For the luciferase reporter assay, the promoter region of *PLIN5* (about 2000 bp upstream of the transcription initiation site of *PLIN5*) was cloned into the digested pGL3-basic vector (digestion sites, KpnI and XhoI).

#### *4.6. Hydrogen Peroxide and LPS Treatment*

The treatment process was the same as in our previous study [12]. Briefly, 30% hydrogen peroxide (i.e., 10 M) was diluted 10,000× by DMEM medium to 1 mM concentration, after the medium had been sterilized using a 0.22 μm filter. The hydrogen peroxide was then diluted to 200 μM and the medium was used to treat cells. The cells were then washed three times using PBS and were treated with different concentrations of hydrogen peroxide media; this operation is important for the treatment of hydrogen peroxide, especially if in low concentrations. Due to the significant impacts of small amounts of metals, such as iron and copper, on the outcomes of in vitro experiments, the medium contained ferric nitrate·9H2O (0.1 mg/L). No other iron or copper was present. The water that was used in this experiment was double distilled and deionized.

#### *4.7. Lipid Droplets Marking and Observation*

The cell slides were fixed with 4% paraformaldehyde for 15 min at room temperature. The slides were stained with BODIPY 493/503 (#D3922, Invitrogen, Carlsbad, CA, USA) for 10 min at 37 ◦C and were then stained with DAPI (#G-1012, Servicebio) for 10 min at 37 ◦C. After washing three times with PBS for 10 min each, the slides were sealed with an anti-fluorescent quenching solution (#P36961, ProLong™ Diamond Antifade Mountant, Invitrogen, Thermo Fisher, USA) for confocal microscopic observation (63× oil lens, BODIPY FL and DAPI channels, Zeiss LSM 800, Germany).

## *4.8. Western Blot*

Western blotting was performed as reported previously [58]. Briefly, cells were collected and homogenized in lysis buffer (#P0013, Beyotime Biotechnology, Nanjing, China). Then, the homogenates were incubated with an SDS-PAGE sample loading buffer (#P0015A, Beyotime Biotechnology, Nanjing, China) at 98 ◦C for 10 min. Subsequently, the samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and were transferred to a polyvinylidene fluoride (PVDF) membrane (Biorad, USA) using a semidry electrophoretic apparatus. The blocked membranes (#P0252-100mL, QuickBlock™ Blocking Buffer for Western Blot, Beyotime Biotechnology, Nanjing, China) were incubated with antibodies overnight at 4 ◦C. The blots were extensively washed three times with tris-buffered saline with tween20 (TBST) buffer for 10 min and were incubated under gentle agitation with the primary antibodies for immunodetection at 37 ◦C for 1.5 h (diluted in QuickBlock™ Primary Antibody Dilution Buffer for Western Blot, #P0256, Beyotime Biotechnology, Nanjing, China). Then, the blots were extensively washed three times with TBST. Subsequently, blots were incubated under gentle agitation with the secondary antibodies for immunodetection at 37 ◦C for 1 h (diluted in QuickBlock™ Secondary Antibody Dilution Buffer for Western Blot, #P0258, Beyotime Biotechnology, Nanjing, China). For detection, M5 eECL Western Blot Kit (#MF-078-01, Mei5bio, Beijing, China) and the chemiluminescence imaging system (LAS4000, ImageQuant, Germany) were used.
