E ab 1034

Immunohistochemistry was used for the identification of p-ERK and EGR1. The protocols were described as follows: blocking buffer: 3% BSA (G5001, Servicebio); primary antibody: anti-p-ERK1/2 antibody (ab201015, Abcam) and anti-EGR1 antibody (22008-1-AP, Proteintech) incubated overnight at 4°C; secondary antibody (E-AB-1034, Elabscience Biotechnology) was incubated for 50 min at room temperature. They were then stained using a DAB Kit (G1211, Servicebio). A light microscope was used for morphology assessment. Average optical density (AOD) values of p-ERK1/2 and EGR1 were analyzed using ImageJ software. AOD = integrated density/area.

Hundred micrograms of protein per lane was subjected to 10% SDS–PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (IPVH00010, Millipore, Darmstadt, Germany). The membranes were blocked with 5% skimmed milk (D8340, Solarbio, Beijing, China) and incubated with primary antibodies against TOM70 (1:1000 dilution, ab89624, Abcam, Shanghai, China; 1:1000 dilution, 14528-1-AP, Proteintech, Wuhan, Hubei, China), TOM40 (1:2000 dilution,18409-1-AP, Proteintech, Wuhan, Hubei, China), TOM22 (1:500 dilution, 11278-1-AP, Proteintech, Wuhan, Hubei, China), TOM20 (1:2000 dilution, 11802-1-AP, Proteintech, Wuhan, Hubei, China), β-actin (1:2000 dilution, 20536-1-AP, Proteintech, Wuhan, Hubei, China), or GAPDH (1:50000 dilution, 60004-1-AP, Proteintech Wuhan, Hubei, China) overnight at 4°C, followed by incubation with secondary species-specific HRP-coupled antibodies (1:5000 dilution, E-AB-1034, Elabscience Wuhan, Hubei, China) for 1 h at room temperature. Proteins were visualized using an ECL chemiluminescence kit (WBKIS0100, Millipore, Darmstadt, Germany) and a chemiluminescence imaging system (ProteinSimple, ChenQ, CA, USA). Bands were quantified using ImageJ software.

AST and ALT testing kits were purchased from Nanjing Jiancheng Biotechnology Institute (Nanjing, China). Lyso PE (18:1) (846725P), CDP-etn (90,756), oleic acid (OA; O1008), and palmitic acid (PA; P0500) were purchased from Sigma (Darmstadt, Germany). An ATP assay kit (S0026), BCA protein assay kit (P0010S), mitochondrial membrane potential assay kit (C2006), and ROS assay kit (S0033S) were obtained from Beyotime (Shanghai, China). RPMI 1640 medium (SH30027.01) and FBS (SH30406.02) were acquired from Gibco (Waltham, MA). PCYT2 (ab135290) antibody was supplied by Abcam (Cambridge); BAX (#5023), Bcl-2 (#3498), cleaved-caspase3 (#9661), and β-actin (#3700) were supplied by Cell Signaling Technology (Danvers, MA); and PISD (sc-390070) was supplied by Santa Cruz Biotechnology (Santa Cruz, CA). Elabscience (Shanghai, China) supplied goat anti-rabbit (E-AB-1034) and goat anti-mouse (E-AB-1035) secondary antibodies. TRIzol reagent (15,596,018) and RevertAid RT kit (K1691) were obtained from Thermo Fisher Scientific (Waltham, MA). The primers for qRT-PCR were synthesized by Sangon Biotech (Shanghai, China). A SYBR green fluorescent probe was obtained from Bio-Rad (Richmond, CA).