2.1 Cells culture

The human cell lines THLE3, Hep3B, Huh7, MHCC97H, Focus, and HA22T were obtained from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences. The cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Cat. No. 11965092, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Cat. No. 10099158, USA), 100 U/mL penicillin (Solarbio), and 100 µg/mL streptomycin (Solarbio). Cultures were incubated at 37 °C in a humidified atmosphere containing 5% CO2.

2.2 Antibodies

Anti-Sp1 rabbit monoclonal (Abcam, Cat No. ab303183, United Kingdom). Mouse anti-Flag mAb (Sigma-Aldrich, Cat No. F1804, St. Louis, USA). Mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mAb (Proteintech, Cat No. 60004-1-Ig, China). Mouse mTOR monoclonal antibody (Proteintech, Cat No. 66888-1-Ig, China). Mouse KAT2A/GCN5 monoclonal antibody (Proteintech, Cat No. 66575-1-Ig, China). Phospho-mTOR (Ser2448) monoclonal antibody (Proteintech, Cat No. 67778-1-Ig, China). HRP-labeled goat anti-mouse IgG antibody (Jackson, Cat No. ab6789, United Kingdom). Anti-succinyllysine mouse mAb (PTM BIO, Cat No. PTM-419, China).

2.3 Bioinformatics analysis website

The StarBase database (https://rnasysu.com/encori/) was utilized to analyze the differential expression of the Sp1 gene in liver hepatocellular carcinoma (LIHC) and normal samples. Specifically, through the Pan-Cancer module, enter the Survival and Differential Expression analysis platform, select “Gene Differential Expression,” input “Sp1,” and choose “LIHC” as the cancer type. The SuccinSite database (http://systbio.cau.edu.cn/SuccinSite/) was employed to predict and analyze the succinylation sites of Sp1. LinkedOmics (http://www.linkedomics.org) and FunRich (http://www.funrich.org/) were used to analyze Sp1-related genes and signaling pathways.

2.4 RNA interference (RNAi), plasmid constructs and transfection

All small interfering RNAs (siRNAs) were designed and synthesized by GenePharma (Shanghai, China). Cells were transfected with the specified siRNAs using Lipofectamine RNAiMAX (Thermo Fisher Scientific, Cat. No. 13778057) according to the manufacturer’s instructions. The transfected cells were then used for subsequent experiments. The genes encoding Sp1, KAT2A, KAT3B, CPT1A, SIRT5, and SIRT7 were synthesized and cloned into the pcDNA3.1 plasmid by GENEWIZ (Suzhou, China), including Sp1 wild-type and related mutant plasmids with a C-terminal Flag tag. Transfections were performed using Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific, Cat. No. 11668019) according to the manufacturer’s protocol.

2.5 RNA extraction and reverse transcription polymerase chain reaction (RT-qPCR)

Adherent cells were seeded in six-well plates and transfected with expression plasmids. Forty-eight hours post-transfection, total RNA was extracted from the cells using RNAiso Plus (TaKaRa, Cat. No. 9108, Japan) according to the manufacturer’s instructions. cDNA was synthesized from the isolated RNA using the PrimeScript RT Reagent Kit (TaKaRa, Cat. No. RR047Q, Japan). The cDNA samples were amplified by quantitative PCR (qPCR) using ChamQ Universal SYBR qPCR Master Mix (Roche, Cat. No. 04913914001) on a LightCycler 480 II (Roche, Basel, Switzerland). Relative mRNA expression levels were quantified using the 2-ΔΔCT method [16], with GAPDH mRNA serving as the endogenous control.

2.6 Western blot (WB)

Cell lysates were prepared by lysing cultured cells with RIPA buffer (Beyotime, Cat. No. P0013B, China) supplemented with a protease inhibitor cocktail (APExBIO, Cat. No. K1007, USA) on ice for 20 min, followed by centrifugation at 12,000 g for 20 min. The supernatants were collected and stored at -80 °C for subsequent use. Proteins were separated on a 10% SDS-PAGE gel and then transferred onto a 0.22-µm PVDF membrane (Merck Millipore, Cat. No. 03010040001, Germany). The membrane was blocked with 5% skim milk in PBS containing 0.05% Tween 20 (PBST) at room temperature for 2 h, then incubated with the appropriate primary antibodies overnight at 4 °C. After washing, the membrane was incubated with HRP-conjugated secondary antibodies at room temperature for 1 h. Immunoreactive bands were detected using an ECL reagent (NCM Biotech, Cat. No. P10300, China) on a chemiluminescence imaging system (Fusion FX7; VILBER, Paris, France).

2.7 Mutation of succinylation sites

Arginine mutations were introduced at K562 (K562R) and K645 (K645R) sites of Sp1. The K562R and K645R plasmids were designed by Genscript Biotechnology Co., LTD (Nanjing, China) and transfected into Hep3B and Huh7 cells for 24 h.

2.8 Immunoprecipitation assay (IP)

Cells were harvested and lysed using WB/IP lysis buffer (Beyotime, Cat. No. P0013B, China) supplemented with a protease inhibitor cocktail (APExBIO, Cat. No. K1007, USA). The lysates were centrifuged at 12,000 g for 20 min, and the supernatants were collected. 10% of the supernatants were reserved as input controls. The remaining lysates were incubated with 20 µL of anti-Flag magnetic beads (MedChemExpress, Cat. No. HY-K0207) overnight at 4 °C. The immune complexes were collected using a magnetic holder and washed with PBS. The complexes were then eluted with 2× SDS loading buffer (TaKaRa, Cat. No. 9173) and subjected to Western blotting using an anti-succinyllysine mouse monoclonal antibody (PTM BIO, Cat. No. PTM-419, China) as the primary antibody and an HRP-labeled goat anti-mouse IgG antibody as the secondary antibody. As for detecting succinylation of Sp1, the total immune complexes were used for western blotting method to obtain the Sp1 protein levels using anti-Sp1 rabbit monoclonal antibody.

2.9 Protein stability assessment

Protein stability assessment was performed to verify the protein stability of Sp1 after KAT2A overexpressing in Hep3B and Huh7 cells. The cells were treated with cycloheximide (CHX, 100 µg/mL, Abcam), a protein translation inhibitor, and the protein level of Sp1 at different time points (0, 6, 12, and 24 h) was detected.

2.10 Cell proliferation evaluation

Cells were seeded into 96-well plates in septuplicate at 1 × 103 per well, and cell viability was tested with a cell counting kit8 (CCK-8) (Solarbio, Cat No. CA1210, China). The absorbance of each well was measured at 450 nm with a Multifunctional microplate reader (Molecular Devices, USA). Cell colony formation was measured fourteen days after cells were seeded into 6 cm dishes. Cell colonies were fixed with 4% paraformaldehyde solution and stained with crystal violet dye. The colony number was calculated. A 5-ethynyl-20-deoxyuridine (EdU) assay kit (Ribobio, Guangzhou, China) was also adopted to inquire the cell proliferation. Cells were seeded into confocal plates with a density of 1 × 106 cells each well, and were incubated with 50 µM EdU buffer at 37 °C for 2 h. Then cells were fixed with 4% formaldehyde for half an hour and permeabilized with 0.1% Triton X-100 for 20 minites. Then the results were visualized by a fluorescence microscope.

2.11 Statistical analysis

Each experiment was conducted with three replicates. Data are presented as the mean ± standard deviation (SD). Statistical analyses were performed using Prism 8.0 software (GraphPad Software, San Diego, USA). The Student’s t-test was used for comparisons between two groups, while one-way ANOVA was used for comparisons among multiple groups. A p-value < 0.05 was considered statistically significant.