Figure 6: K81 mutations promote tumour cell growth in vitro and in vivo.

(a) Verification of HepG2 stable cell lines. Knockdown efficiency and re-expression levels of wild-type or K81R/Q mutants were determined by western blotting. (b) K81R and K81Q mutations reverse the proliferative disadvantage of HepG2 cells upon folate deprivation. HepG2 stable cell characterized in a were cultured in normal or folate-deprived medium. Direct cell count was performed every 24 h after seeding. Error bars represent cell numbers±s.d. for triplicate experiments. The two-tailed Student’s t-test was used. **P<0.01; ***P<0.001. (c,d) K81R and K81Q mutants promote xenograft tumour growth. Subcutaneous xenograft experiment was performed in nude mice using HepG2 stable cells. Major and minor diameters of tumours were measured and tumour volumes were calculated. The two-tailed Student’s t-test was used. *P<0.05; **P<0.01; ***P<0.001; NS denotes no significance (c). 25 days after injection, tumours were dissected, photographed and weighted. The two-tailed Student’s t-test was used. **P<0.01; NS denotes no significance (d). (e) The expression of wild-type, K81R and K81Q MAT IIα in xenografts. Whole-cell lysates were prepared from either original HepG2 stable cell lines or xenograft tumours, followed by western blotting analysis. (f) The hepatocellular cancer clinical samples show an inverse correlation between MAT IIα protein and K81 acetylation. Human hepatocellular cancer samples each paired with cancerous tissue (designated as C) and adjacent normal tissue (designated as N) were lysed and directly subjected to western blotting. Only eight pairs of samples showcasing inverse correlation are shown. For more samples, please refer to supplementary Fig. 6f. (g) Working model. By enhancing MAT IIα association with P300 and UBR4, folate deprivation promotes both MAT IIα K81 acetylation and ubiquitylation, resulting in its proteasomal degradation. Folate stabilizes MAT IIα by dissociating MAT IIα-P300-UBR4 complex, and promoting MAT IIα–HDAC3 interaction. Accumulated MAT II α protein facilitates rapid cell proliferation and, therefore, promotes tumour growth.