Fig. 2: USP22 promotes lipid accumulation and tumorigenesis in HCC cells.

a Venn diagram of the overlap between USP22-knockdown (shUSP22-1 vs shControl) and USP22-overexpression (USP22 vs Control)-induced differential metabolites (The heat map of differential metabolites is shown in Supplementary Fig. 3d and 3e, p < 0.05, unpaired two-tailed Student’s t-test). The red number 34 refers to the number of metabolites with the opposite trend. LC-MS-based nontargeted metabolomic analysis, and the data were corrected by total peak area. b Heatmap analysis of these 34 significantly changed metabolites. p < 0.05, unpaired two-tailed Student’s t-test. Red indicates increase, and blue indicates decrease. -1.5~1.5 indicates the Fold Change. c Enriched signaling pathways identified by pathway analysis based on these 34 differential metabolites. (https://www.metaboanalyst.ca/). d The percentages of various isotopomers of FA 16:0 (palmitate) after trace to [U-¹³C] glucose in MHCC-97H-shUSP22-1-and MHCC-97L-USP22cells. Medium was changed to RPMI 1640 containing [U-¹³C] glucose (2 g/L) when the cell density was about 80%, and 24 h later cell culture plates were washed with PBS and snap-frozen in liquid nitrogen and subjected to LC-MS analysis. unpaired two-tailed Student’s t-test. The data shown represent the means (±SD) of biological replicates. The experiments were repeated four times (n = 4). e Representative images of Oil red staining assay in MHCC-97H-shUSP22-1/2 cells and MHCC-97L-USP22 cells. Cells were analyzed after 24 h adherence. Scale bars, 50 μm. f Relative content of TG was analyzed in MHCC-97H-shUSP22-1/2 cells and MHCC-97L-USP22 cells. Cells were analyzed after 24 h adherence. The data shown represent the means (±SD) of biological replicates. The experiments were repeated five times (n = 5). One-way ANOVA test. g–i MHCC-97H-shUSP22-1 or shControl cells were injected into the right flank of nude mice. Tumor volumes were measured every 3 days. Tumor images (g), growth curves (h) and weight (i) were obtained at day 21 after dissection. Data in h are presented as mean values ± SD and data in i are presented as mean values with minima and maxima. unpaired two-tailed Student’s t-test. Scale bars, 1 cm. n = 6 biologically independent tumor samples. j Heatmap analysis of significantly changed metabolites in MHCC-97H-shUSP22-1 cells-derived tumors compared to shControl cells-derived tumors based on nontargeted metabolomic analysis. Data were standardized by total peak area. p < 0.05, unpaired two-tailed Student’s t-test. Source data are provided in the Source Data file.