Fig 2: The expression of the Trx system in pan-cancer. (A) The expression of selected genes in pan-cancer based on TCGA and GTEx database. (B) Immunohistochemical images of Trx system proteins in tumor and normal tissues of gastric cancer, lung cancer and colorectal cancer from HPA database. (C) The protein expression levels of TXN, TXNIP and TXNRD1 based on CPTAC database.

Fig 3: Auranofin enhances the sensitivity to sorafenib-induced apoptosis via inhibition of TXNRD1 in PCK1-KO cells in vitro. (A) Cell proliferation curves. PCK1-KO hepatoma cells were treated with 0.6 μmol/L auranofin or 5 μmol/L sorafenib alone or in combination. Cells were plated into 96-well plates at 1 × 103 cells/well and were counted every 24 h in triplicate. Data are presented as the mean ± SD; *P < 0.05 vs. Sorafenib alone. (B) ROS were monitored by fluorescent labeling (red) and nuclei were stained by DAPI (blue) in cells treated with sorafenib and auranofin alone or in combination. Scale bar = 10 μm. (C) Protein expression of Caspase3/9, Cleaved caspase3/9, Bax, Bcl2, TXNRD1, and PCK1. PCK1-KO hepatoma cells were treated as in Figure 4A for 48 h and the proteins were isolated and analyzed by western blotting. GAPDH expression served as an internal control. (D) Flow-cytometric analysis of apoptosis was performed after treatments with sorafenib and/or auranofin. (E) Quantification of cell death detected in Figure 4C. Cell apoptosis as detected by PI staining. Values represent the mean percentage of dead cells ± SD from three samples (*P < 0.05,***P < 0.001). (F) Schematic illustration showing the PCK1–NADPH–ROS-TXNRD1 axis. In HCC, decreased PCK1 suppressed the generation of NADPH via pentose phosphate pathway (PPP). Subsequently, ROS, which is quenched by NADPH, was increased, further promoted nuclear translocation of Nrf2 and the expression of TXNRD1. The cropped blots are used in the figure and full length blots are presented in Figure S6.

Fig 4: PCK1 represses TXNRD1 expression in hepatoma cells and hepatic tissues. (A) Volcano plot of RNA-seq data from Huh7 cells infected with adenoviruses expressing PCK1 (AdPCK1) vs. Huh7 cells infected vector control (AdGFP). In total, 498 genes were differentially expressed (fold change >1.5 or < 0.667; FDR < 0.05). TXNRD1 is downregulated in PCK1-overexpressing hepatoma cells. (B) RT-qPCR analysis of mRNA levels of TXNRD1 in Huh7 cells infected with AdPCK1 or AdGFP. Total mRNA was isolated at 36 h after infection. Elevated mRNA expression of TXNRD1 in PCK1-KO (endogenous PCK1 was knocked out in PLC/PRF/5 cells by CRISPR-Cas9) compared to parental cells. Data are shown as the mean ± SD (n = 3 independent experiments; **P < 0.01 by Student's t-test). (C) Western blot analysis of TXNRD1 in Huh7 cells infected with AdPCK1 or AdGFP. (D) Protein levels of TXNRD1 were detected by western blot analysis in PCK1-KO cells and parental cells. (E) PCK1 and TXNRD1 protein levels in eight cases of paired primary hepatocellular carcinoma tissues and adjacent non-tumor tissues. GAPDH was used as a loading control. The cropped blots are used in the figure and full length blots are presented in Figure S3.

Fig 5: PCK1 inhibits tumor growth and oxidative stress in xenograft tumors. MHCC-97H cells were infected with AdGFP or AdPCK1 or mock-infected for 24 h, then subcutaneous injected into nude mice (n = 6). (A) Representative image of tumor in all groups (presented two tumors each group). (B) The growth curve of subcutaneous xenograft tumor. Tumor volumes were measured every 4 days and calculated as length × (square of width)/2 (repeated-measures analysis of variance, *P < 0.05 compared to AdGFP group, error bars represent mean ± SD, n = 6). (C) Tumor size of subcutaneous xenografts after dissected. (D) ROS monitored by immunofluorescent labeling (red) and nuclei by DAPI (blue) staining in xenografts. Scale bar = 200 μm. (E) Expression of PCK1 and TXNRD1 checked by western blot assay in xenograft tumor samples. Semi-quantitative densitometry graphs of immunoblots (arbitrary densitometry units) for PCK1 and TXNRD1 levels normalized to β-actin expression are shown. (*P < 0.05, **P < 0.01, AdPCK1 group vs. AdGFP group). The cropped blots are used in the figure and full length blots are presented in Figure S5.