Fig 2: SLFN11 is downregulated in human HCC and correlates with poor prognosis. (A) qRT-PCR analysis of SLFN11 mRNA expression in 116-paired tumor and nontumor liver tissues. (B, C) Western blot of SLFN11 protein expression in 12-paired nontumor (N) and tumor (T) liver tissues. (D) mRNA and protein expression level of SLFN11 in a normal liver cell line (L-02)and four HCC cell lines (HCCLM3, Hep3B, SMMC-7721, and PLC/PRF/5). (E) Representative IHC staining images indicating low and high expression of SLFN11 in HCC tissue microarray. Scale bars = 200 µm or 20 µm, respectively. (F) Kaplan-Meier curves for overall survival and recurrence-free survival based on SLFN11 expression in the Fudan LCI cohort 1. *** P < 0.001.

Fig 3: SLFN11 inhibits HCC progression in vivo, as well as the proteomic analyses of SLFN11 complexes by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). (A) Effects of SLFN11 overexpression on HCC progression by establishment of subcutaneous xenograft mouse models. Tumor growth curves and weight of xenografts derived from HCCLM3-Vector or HCCLM3-SLFN11 cells are shown. (B) Effects of SLFN11 knockdown on HCC progression by establishment of subcutaneous xenograft mouse models. Tumor growth curves and weight of xenografts derived from SMMC-7721-Control or SMMC-7721-shSLFN11 cells are shown. (C) Venn diagram shows the overlapping and unique proteins identified from the complexes related to SLFN11 overexpression and the vector control cells. (D) Functional enrichment analysis of the unique proteins related to SLFN11 with ingenuity pathway analysis (IPA) software. (E) Pathway enrichment analysis of the unique proteins related to SLFN11 with IPA software. (F) The peptide spectrum of RPS4X by LC-MS/MS assay. The N-terminal and C-terminal collision-induced dissociation fragment ions are indicated by b and y, respectively. ** P < 0.01, *** P < 0.001. Abbreviations: QE, Q Exactive.

Fig 4: SLFN11 inhibits tumor metastasis in vivo. (A, B) Left panel: Representative hematoxylin and eosin (HE) staining images indicate the effects of SLFN11 and INK128 on lung metastasis from orthotopic nude mice in HCCLM3 and SMMC-7721 cells. Scale bars = 1 mm and 200 µm. Right panel: Histograms show metastatic nodules in the lungs with a mean ± SD in each group. ** P < 0.01, *** P < 0.001. (C) A schematic illustrating the role of SLFN11 in regulating HCC tumorigenesis and metastasis.

Fig 5: RPS4X is an essential factor in SLFN11-mediated inhibition of the mTOR signaling pathway. (A) Western blot of the knockdown efficiency of RPS4X in HCCLM3 cells. (B) Western blot indicates that once RPS4X was knocked down in HCCLM3 cells, regardless of whether SLFN11 was overexpressed, the phosphorylation of S6 and eIF4E were inhibited at almost the same level. (C) Colony formation assays were conducted to study cell proliferation of HCCLM3-VectorControl cells and HCCLM3-SLFN11 cells with or without RPS4X knockdown. (D) Wound healing assays were performed to detect cell migratory abilities of HCCLM3-VectorControl cells and HCCLM3-SLFN11 cells with or without RPS4X knockdown. (E) Transwell assays were used to investigate the cell migratory and invasive capacities of HCCLM3-VectorControl cells and HCCLM3-SLFN11 cells with or without RPS4X knockdown. (F) CCK-8 assays were conducted to determine the cell proliferation of HCCLM3-VectorControl cells and HCCLM3-SLFN11 cells with or without RPS4X knockdown. (G) Cell apoptosis was assessed by flow cytometry in HCCLM3-VectorControl cells and HCCLM3-SLFN11 cells with or without RPS4X knockdown. ** P < 0.01, *** P < 0.001, NS, not significant.