Fig 2: Glycine decarboxylase (GLDC) overexpression inhibits intrahepatic metastasis in vivo.a Representative images showing the orthotopic transplanted tumor (marked with bold blank arrows) and the intrahepatic metastases (marked with thin blank arrows) in the orthotopic mouse models transplanted with GLDC-overexpressing HCCLM3 cells and the corresponding control cells. b Histogram showing the surface tumor nodules (n = 5, *P < 0.05, by Student’s t-test). c Representative hematoxylin and eosin (H&E) staining images of the orthotopic transplanted tumor (marked with bold blank arrows) and the intrahepatic metastasis (marked with bold yellow arrows). d Representative immunohistochemistry (IHC) images of expressions of p62 in the orthotopic transplanted tumors transplanted with GLDC-overexpressing HCCLM3 cells and the corresponding control cells

Fig 3: Glycine decarboxylase (GLDC) regulates migration and invasiveness through epigenetic silencing by miR-30d-5p.a Transwell chamber assays using Huh7 cells co-transfected with miR-30d-5p mimics and GLDC expression construct. b Representative images of the migratory cells (left panel), magnification: ×200. Histogram of the numbers of migratory (right panel, *P < 0.05). c Matrigel invasion assays using Huh7 cells co-transfected with miR-30d-5p mimics and GLDC expression construct. Representative images of the invading cells (left panel), magnification: ×200. d Histogram of the numbers of invasion (right panel, *P < 0.05)

Fig 4: RT-qPCR and western blotting validation of the expression level of SUCLG1, PCK2, GLDC in OSRC-2 and A498 cell lines. a–c Forty-eight hours after plasmid or vector transfection, qPCR detected the expression level of 3 genes in both OSRC-2 and A498 cell lines. b–f Protein expression was evaluated by Western blot. j–l The values of the band intensity represent the densitometric estimation of each band normalised by β-actin in (b–f, respectively). (*p < 0.05 and **p < 0.01)

Fig 5: RT-qPCR and western blotting of the expression level of key genes in RCC cell lines. a–f RT-qPCR detection of mRNA expression of SUCLG1, PCK2, GLDC, SLC12A1, ATP1A1, PDHA1 in seven RCC cell lines. g Key genes mRNA expression level in eight paired kidney tumor tissues. h SUCLG1, PCK2, GLDC protein expression level in eight paired kidney tumor tissues (T) and their adjacent normal tissues (ANT). i The values of the band intensity represent the densitometric estimation of each band normalised by β-actin in (H). (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.001)