Fig 2: CS increases the stemness of hESCs in vivo. (a) Images of immunohistochemistry staining of CK19 and ITGβ1 at day 10 after surgery. CK19 positive cells were around the hair follicles and stained in brown. ITGβ1 positive cells were inside the epidermis and stained in brown. Scale bar: 20 μm. (b) The expression level of ITGβ1 and CK19. (c) The relative expression intensity of ITGβ1 and CK19 in control and CS (+) groups of the immunohistochemistry staining. **p < 0.01. (d) The relative protein level in control and CS (+) groups. The results were normalized to GAPDH expression. *p < 0.05; **p < 0.01. CK19 cytokeratin 19, CS calcium silicate, GAPDH glyceraldehyde 3-phosphate dehydrogenase, hESCs human epidermal stem cells, ITGβ1 integrin beta-1

Fig 3: Double immunofluorescence staining of p-ERK in livers with sLC and nLC at 0 h and 6 h NMP. The p-ERK signal is represented in red; the hepatocytes (labeled with hepatocyte specific antigen (Hep PAR-1) and cholangiocytes (labeled with CK-19) are represented in green; the nuclei are represented in blue (DAPI). CK-19, cytokeratin-19; DAPI, 4′,6-diamidino-2-phenylindole; p-ERK, phosphorylated extracellular signal-regulated kinase; nLC, nonsuccessful lactate clearance; NMP, normothermic machine perfusion; sLC, successful lactate clearance.

Fig 4: Identification of hESCs and the effects of CSIEs dissolution products on hESCs proliferation. (a) Flow cytometry demonstrated that the cells expressed a positive phenotype for CK19 and ITGβ1. (b) The statistical analysis of CCK-8 assay that reveals hESCs viability in four concentrations of CSIEs dilutions and EpiLife medium as a control (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001. (c) Flow cytometric analysis of cell cycle. (d) The percentage of hESCs in the G0/G1, S and G2/M phases, respectively (n = 5). (e) EdU positive cells (red) and nuclei (blue) in 1/64, 1/128, 1/256 CSIEs dilutions and EpiLife medium. Scale bar: 50 μm. (f) The statistical analysis of the percentage of EdU positive cells (n = 5). *p < 0.05, ***p < 0.001. CCK-8 cell counting kit-8, CK19 cytokeratin 19, CSIEs calcium silicate ion extracts, EdU 5-ethynyl-2′-deoxyuridine, G1 first gap, G2 second gap, hESCs human epidermal stem cells, ITGβ1 integrin beta-1, M mitosis, OD optical density, S synthesis

Fig 5: DDX3 suppresses stemness gene signature.(a) Lower expression of DDX3 along with overexpression of stemness markers was observed in the poorly differentiated cell line. Cell lysates (50 μg) of HepG2, Hep3B, HuH-7 and SK-Hep-1 cells were analyzed by western blotting with antibodies against DDX3, Nanog, Oct4, c-Myc, Sox2, KLF4, Bmi1, CK19 and β-actin. (b) Knockdown of DDX3 led to up-regulation of stemness markers. Cell lysates (50 μg) of stable shLuc, shDDX3 #2 and shDDX3 #3 HepG2 cells were analyzed by western blotting with antibodies described in (a). (c) DDX3 overexpression suppressed stemness markers. Plasmid pcDNA3-SRα/FLAG or pcDNA3-SRα/FLAG-DDX3 was transfected into SK-Hep-1 cells. At 48 h post transfection, cell lysates were prepared and subjected to immunoblotting with antibodies described in (a) and anti-FLAG antibody. β-actin was used as internal control in (a–c). (d) DDX3 knockdown was correlated with up-regulation of hepatic CSC surface markers. mRNA expressions of DDX3, CD133, CD13, EpCAM, CD90 and GAPDH in shLuc, shDDX3 #2 and shDDX3 #3 cells were detected by qRT-PCR. GAPDH was used as internal control. Fold change of each mRNA transcript in shDDX3 #2 and shDDX3 #3 cells was relative to that of shLuc cells. (e) DDX3 overexpression resulted in suppression of hepatic CSC surface markers. SK-Hep-1 cells were transfected with plasmid pcDNA3-SRα/FLAG or pcDNA3-SRα/FLAG-DDX3 as described in (c). At 48 h post transfection, total RNA was extracted and subjected to qRT-PCR analysis. GAPDH was used as internal control. Fold change of each mRNA transcript in FLAG-DDX3-expressing cells was relative to that of vector control cells. All experiments were performed at least three times, and the error bar indicates ± 1 s.d. of the mean. Statistical analyses were carried out using t test (*p < 0.05; **p < 0.01; ***p < 0.001).