Fig. 1: MTR4 is required for the tumorigenesis of HCC cells.

a Heat map of the global mRNA expression profile in non-tumor tissues (n = 220) and hepatocellular carcinoma (HCC) tissues (n = 225) in GSE14520 dataset. The levels of MTR4 are indicated with a white line. b Box plot showing the relative mRNA levels of MTR4 in HCC tissues (n = 225) and non-tumor tissues (n = 220) in GSE14520 (GPL3921) dataset. The significance of the difference was assessed by two-tailed, unpaired t-test. p value is indicated. Centre is median within box, bound of the box spans the interquartile range, and whiskers visualize 5 and 95% of the data points. c, d Chips of HCC samples (n = 108) and adjacent non-tumor (ANT) samples (n = 108) were stained with anti-MTR4 antibody and the intensity of staining was scanned and scored. Representative immunohistochemistry (IHC) images are shown. Two-tailed, unpaired t-test. Data are presented as mean value ± s.d. p value is indicated. Scale bar = 200 µm. e The MTR4 mRNA levels in HCC samples (n = 77) were compared with the corresponding ANT samples (n = 77). Eighty-three percent (64/77) of HCC samples have higher MTR4 mRNA levels than ANT. f The MTR4 levels were inversely correlated with the postoperative recurrence-free survival (RFS) of HCC patients. RFS of the patients with high MTR4 mRNA levels (n = 12) is significantly lower than those with lower MTR4 (n = 50). The difference in survival rates was assessed with the log-rank test (Mantel Cox). p value is indicated. g The knockdown of MTR4 in HCC cells PLC/PRF/5 was confirmed by western blotting. CTL, HCC cells expressing scramble shRNA. Tubulin was used as an internal control. Consistent data were obtained from two independent experiments. h Proliferation of MTR4 KD and control cells was analyzed with CCK8 assay. n = 3 biologically independent experiments. Difference between two groups was calculated by two-way ANOVA, followed by Bonferroni post-tests. Data are presented as mean value ± s.d. p value is indicated. i Colony formation assay of the control and MTR4 KD cells. Difference between two groups was calculated by two-tailed, unpaired t-test. Data are presented as mean value ± s.d. p value is indicated. n = 3 biologically independent samples. j Inducible knockdown of MTR4 in PLC/PRF/5 cells (iMTR4 cells) was confirmed by western blotting after the treatment with 1 µg/ml doxycycline (Doxy) for 4 days. Consistent data were obtained from two independent experiments. k, l The volumes (k) and weight (l) of tumors formed by PLC/PRF/5 cells expressing scramble shRNA (iSC) or iMTR4 cells in NSG mice were measured after daily i.p. injection of Doxy (20 mg kg⁻¹ body weight) or mock treatment for 8 days. Individual tumor volumes were measured every day after doxy treatment. Repeated measures two-way ANOVA, followed by Turkey’s post-tests. p value is indicated. Repeated measures two-way ANOVA, followed by Bonferroni post-tests. Data are presented as mean value ± s.d. p value is indicated. n = 6 independent samples for each group. At the end of the treatment, the weight of all tumors in each group was compared. Mann–Whitney test. p value is indicated. n = 6 independent samples for each group. Source data are provided as a Source Data file.