Fig 2: GRAMD4 recruited the E3 ligase ITCH to target TAK1 for K48-linked ubiquitination and degradation. (A) 293T cells were transfected with vector or GRAMD4-Flag plasmid together with control-siRNA or ITCH-siRNAs and analysed for TAK1, ITCH and Flag-tag expression by western blot. (B) HLF cells with vector and GRAMD4 overexpression were transiently transfected with control-siRNA or ITCH-siRNAs and analysed for TAK1, ITCH and GRAMD4 expression by western blot. (C) Co-IP analysis of the binding between exogenous GRAMD4-Flag and ITCH-HA in co-transfected HEK293T cells. (D) Immunoassay of lysates from HLF cells, followed by immunoprecipitation with an anti-TAK1 antibody. Co-IP assay of binding between TAK1 and ITCH was performed at exogenous (E) endogenous (F) levels when GRAMD4 was overexpressed or not. Immunoassay of 293T cells transfected with expression vectors for various combinations of AMD4-Myc, TAK1-Flag, HA-Ub (WT)(G), Ub (K48)(H) and Ub (K63)(I) together with control-siRNA or ITCH-siRNA, followed by immunoprecipitation of lysates with an anti-Flag antibody and immunoblot analysis with anti-Myc, anti-HA, anti-Flag anti-ITCH antibodies

Fig 3: GRAMD4 and TAK1 had an inverse correlation in HCC tissues. (A) Western blot analysis of GRAMD4 and TAK1 expression in HCC and non-cancerous tissues, GAPDH was used as a loading control. (B) Correlation analysis of GRAMD4 and TAK1 expression according to western blot analysis in HCC tissues from the investigated HCC patients. (C) IHC staining of GRAMD4 and TAK1 expression in HCC tissues from clinical HCC samples. The images are representative of figures from the investigated HCC patients (Scale bar: 250 µm, 25 µm). (D) Correlation analysis of GRAMD4 and TAK1 expression according to IHC staining in HCC tissues from the investigated HCC patients. (E-F) Kaplan–Meier analysis of the overall and disease-free survival of HCC patients stratified by GRAMD4/TAK1 expression levels. Statistical analysis was performed using Pearson's correlation in (B, D). (G) A proposed model for the mechanism through which GRAMD4 downregulates TAK1 by recruiting ITCH, resulting in the deactivation of the MAPK signalling pathway and inhibition of HCC progression and metastasis

Fig 4: GRAMD4 expression was downregulated in HCC and predicted poor clinical outcomes. (A) GRAMD4 mRNA expression in HCC tissues and adjacent normal samples extracted from GSE22058, GSE14520, GSE63898 and TCGA. (B) GRAMD4 CNV status in HCC from TCGA. (C) Representative immunohistochemical staining images of GRAMD4 in 110 paired HCC tumour tissues and adjacent non-tumour tissues (Scale bar: 250 µm, 25 µm). (D) Statistical analysis of GRAMD4 expression according to the IHC assay in HCC tissues and matched adjacent tissues. (E) Univariate regression analysis of the relation between the GRAMD4 and clinicopathological characteristics regarding OS in the Tongji cohort. Overall survival (F) and disease-free survival (G) of HCC patients with low or high tumour GRAMD4 scoring in the Tongji cohort. Overall survival (H) of HCC patients with low or high GRAMD4 expression in the TCGA cohort

Fig 5: GRAMD4 interacted and co-localized with TAK1. (A) MS analysis of GRAMD4-associated proteins. (B) Co-IP analysis of the binding between exogenous GRAMD4-Flag and TAK1-HA in co-transfected HEK293T cells. (C-D) Co-IP analysis of the binding between endogenous GRAMD4 and TAK1 in HLF and Hep3B cells. (E) HLF cells were fixed and stained with TAK1 antibody (Red) and GRAMD4 antibody (Green). Nuclei were counterstained with DAPI (blue). Scale bar: 20µm. (F) Schematic illustration showing the wild-type and truncation mutants of GRAMD4. (G) Co-IP analysis of the interaction between TAK1 and full-length GRAMD4 or its truncation mutants in HEK293T cells co-transfected with TAK1-HA plasmid and GRAMD4-Flag plasmid or GRAMD4-Flag truncation mutant plasmids