Journal: Stem Cell Research & Therapy

Article Title: Isolation and characterisation of AFP + /DLK1 + double-positive hepatic stem/progenitor-like cells from fibrotic partial hepatectomy model

doi: 10.1186/s13287-025-04728-1

Figure Lengend Snippet: Schematic presentation of the strategy for isolating AFP + /DLK1 + double-positive cells from the fibrotic PHx model

Article Snippet: Cells were then blocked with 2.5% BSA for 1 h. The cells were subsequently incubated with primary antibodies against AFP (1:200; Proteintech-cn, Wuhan, 14550-1-AP) and DLK1 (1:200; MBL, Beijing, D187-3) at 4 °C overnight, followed by incubation with secondary antibodies for 1 h at room temperature in the dark (1:1000, Abcam, USA).

Techniques:

Journal: Stem Cell Research & Therapy

Article Title: Isolation and characterisation of AFP + /DLK1 + double-positive hepatic stem/progenitor-like cells from fibrotic partial hepatectomy model

doi: 10.1186/s13287-025-04728-1

Figure Lengend Snippet: Pre-injection of CCl 4 promotes liver regeneration after 2/3 PHx. a Schematic drawing of mouse liver anatomy and positioning of silk threads for knots. b Liver /Body Weight Ratio (%) after PHx. n = 2 per group. c Level of serum ALT and AST after PHx. n = 3 per group. d Relative mRNA expression levels of Afp , Dlk1 , Lgr5 , Ki67 , HNF4α , Alb , Krt-8 , Krt-18 , Krt7 ,and Krt-19 in regenerating livers after PHx. Values are normalised to Actb. n = 3 per group. e AFP (green), DLK1 (red), and DAPI (blue) immunofluorescence analyses in regenerating livers after PHx on the fibrotic model. Scale bars: 100 μm. f Relative fluorescence intensity (%). Statistical analysis was undertaken with Two-way ANOVA; (* p < 0.05; ** p < 0.01; *** p < 0.001, **** p < 0.0001.)

Article Snippet: Cells were then blocked with 2.5% BSA for 1 h. The cells were subsequently incubated with primary antibodies against AFP (1:200; Proteintech-cn, Wuhan, 14550-1-AP) and DLK1 (1:200; MBL, Beijing, D187-3) at 4 °C overnight, followed by incubation with secondary antibodies for 1 h at room temperature in the dark (1:1000, Abcam, USA).

Techniques: Injection, Expressing, Immunofluorescence, Fluorescence

Journal: Stem Cell Research & Therapy

Article Title: Isolation and characterisation of AFP + /DLK1 + double-positive hepatic stem/progenitor-like cells from fibrotic partial hepatectomy model

doi: 10.1186/s13287-025-04728-1

Figure Lengend Snippet: Isolation of AFP + /DLK1 + Double-Positive Cells from regenerating liver by density gradient differential centrifugation. a Program diagram of density gradient differential centrifugation. Whole liver cells obtained by enzymatic digestion (as shown in ①). By lowspeed centrifugation (50× g , 5 min), liver parenchymal cells are deposited at the bottom of the centrifuge tube, and liver nonparenchymal cells are suspended in the supernatant (as shown in ②). The supernatant is collected and centrifuged at medium speed (200× g , 5 min) to obtain nonparenchymal cell masses (as shown in ③). The nonparenchymal cells are placed in the top layer of the gradient centrifuge solution (as shown in ④). By differential centrifugation (600× g , 10 min, sped up by 9, and sped down by 1), all kinds of cells are separated into different Percoll layers (as shown in ⑤). Cells are collected from 50%–70% Percoll layer and HSPClike cells obtained by centrifugation (250× g , 5 min) (as shown in ⑥). b Flow cytometry analysis of AFP + /DLK1 + double-positive cells in each Percoll layer. c Percentage of AFP + /DLK1 + double-positive cells (by flow cytometry). d Relative mRNA expression levels of Afp , Dlk1 , Lgr5 , and Ki67 in each Percoll layer cells. Values are normalised to Actb. n = 3 per group. e AFP (green), DLK1 (red), and DAPI (blue) immunofluorescence analyses in the 50%– 70% layer. Scale bars:20 μm. f Relative fluorescence intensity (%). Statistical analysis was undertaken with Two-way ANOVA; (* p < 0.05; **** p < 0.0001.)

Article Snippet: Cells were then blocked with 2.5% BSA for 1 h. The cells were subsequently incubated with primary antibodies against AFP (1:200; Proteintech-cn, Wuhan, 14550-1-AP) and DLK1 (1:200; MBL, Beijing, D187-3) at 4 °C overnight, followed by incubation with secondary antibodies for 1 h at room temperature in the dark (1:1000, Abcam, USA).

Techniques: Isolation, Centrifugation, Gradient Centrifugation, Flow Cytometry, Expressing, Immunofluorescence, Fluorescence

Journal: Discover Oncology

Article Title: AFP confers the resistance of lenvatinib in hepatocellular carcinoma by activating PI3K/AKT/LDHA signaling axis

doi: 10.1007/s12672-026-04637-6

Figure Lengend Snippet: Expression of AFP and LDHA in HCC patient tissues and their correlation. a Microarray matrix of tumor tissues and corresponding adjacent tissues from 30 HCC patients. b Immunohistochemistry images of AFP expression in tumor tissues versus adjacent tissues (left); Statistical analysis of AFP expression differences between tumor and adjacent tissues (right). c Immunohistochemistry images of LDHA expression in tumor tissues versus adjacent tissues (left); Statistical analysis of LDHA expression differences between tumor and corresponding adjacent tissues (right); Scale bar in magnified images represents 50 µm; * P < 0.05, indicating statistically significant differences. d Correlation between AFP and LDHA in HCC patient tissues analyzed by bivariate correlation using SPSS 26.0, with correlation coefficient r 2 = 0.7162, P < 0.0001

Article Snippet: Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum were purchased from Gibco (USA); 0.25% trypsin digestion solution was obtained from Beyotime Biotechnology (Shanghai, China); lenvatinib and LY294002 inhibitors were purchased from MedChemExpress(USA); AFP, HK2, LDHA, PI3K, and AKT antibodies were acquired from Abcam (USA); PKM2, P-gp, Bcl-2, and PFK1 antibodies were purchased from Proteintech(USA); and glucose and lactic acid test kits were obtained from Nanjing Jiancheng Bioengineering Institute(Nanjing, China).

Techniques: Expressing, Microarray, Immunohistochemistry

Journal: Discover Oncology

Article Title: AFP confers the resistance of lenvatinib in hepatocellular carcinoma by activating PI3K/AKT/LDHA signaling axis

doi: 10.1007/s12672-026-04637-6

Figure Lengend Snippet: The effects of AFP on the proliferation and drug resistance of liver cancer cell lines. a The efficiency of lentiviral infection in cells was observed under fluorescence microscopy; Western blotting analysis was performed to examine the expression of AFP before and after transfection in cells. Images were captured at 100 × magnification, with a scale bar length of 100 µm. b Different concentrations of lenvatinib were applied to three groups of cells: HuH-7, HuH-7-shNC, and HuH-7-shAFP, as well as HLE, HLE-NC, and HLE-AFP. After 6 days, the cell inhibition rate was evaluated using the MTT assay. c Cells were treated with 10 µM lenvatinib, and colony formation assays were conducted to assess proliferation. * P < 0.05, ** P < 0.01. N = 3

Article Snippet: Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum were purchased from Gibco (USA); 0.25% trypsin digestion solution was obtained from Beyotime Biotechnology (Shanghai, China); lenvatinib and LY294002 inhibitors were purchased from MedChemExpress(USA); AFP, HK2, LDHA, PI3K, and AKT antibodies were acquired from Abcam (USA); PKM2, P-gp, Bcl-2, and PFK1 antibodies were purchased from Proteintech(USA); and glucose and lactic acid test kits were obtained from Nanjing Jiancheng Bioengineering Institute(Nanjing, China).

Techniques: Infection, Fluorescence, Microscopy, Western Blot, Expressing, Transfection, Inhibition, MTT Assay

Journal: Discover Oncology

Article Title: AFP confers the resistance of lenvatinib in hepatocellular carcinoma by activating PI3K/AKT/LDHA signaling axis

doi: 10.1007/s12672-026-04637-6

Figure Lengend Snippet: The effect of AFP on liver cancer cell apoptosis under lenvatinib treatment. a Apoptosis of HuH-7 cells without treatment and with 10 µM lenvatinib before and after AFP expression interference; apoptosis of HLE cells without treatment and with 10 µM lenvatinib before and after AFP overexpression, detected by TUNEL assay. b Cell viability assessed by trypan blue staining, where white cells indicated by black arrows represent live cells and blue cells indicated by red arrows represent dead cells. c Western blotting analysis of apoptosis-related protein expression, the below bar chart represents the statistical analysis of the gray-scale scan values of protein bands. Images were captured at 200 × magnification, with a scale bar length of 50 µm; * P < 0.05, ** P < 0.01. N = 3

Article Snippet: Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum were purchased from Gibco (USA); 0.25% trypsin digestion solution was obtained from Beyotime Biotechnology (Shanghai, China); lenvatinib and LY294002 inhibitors were purchased from MedChemExpress(USA); AFP, HK2, LDHA, PI3K, and AKT antibodies were acquired from Abcam (USA); PKM2, P-gp, Bcl-2, and PFK1 antibodies were purchased from Proteintech(USA); and glucose and lactic acid test kits were obtained from Nanjing Jiancheng Bioengineering Institute(Nanjing, China).

Techniques: Expressing, Over Expression, TUNEL Assay, Staining, Western Blot

Journal: Discover Oncology

Article Title: AFP confers the resistance of lenvatinib in hepatocellular carcinoma by activating PI3K/AKT/LDHA signaling axis

doi: 10.1007/s12672-026-04637-6

Figure Lengend Snippet: The influence of the PI3K/AKT signaling pathway in AFP-regulated LDHA-mediated Warburg effect progression. a KEGG pathway enrichment analysis of AFP-related genes in HCC, showing a significant positive correlation between AFP and the PI3K/AKT signaling pathway ( P = 0.035). b – e Western blotting analysis of changes in PI3K/AKT signaling pathway and glycolysis-related protein expression in HuH-7 cells before and after interfered the expression of AFP, as well as in HLE cells before and after AFP overexpression, following treatment with the PI3K/AKT pathway inhibitor LY294002 for 12 h, the right bar chart represents the statistical analysis of the gray-scale scan values of protein bands. * P < 0.05, ** P < 0.01. N = 3

Article Snippet: Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum were purchased from Gibco (USA); 0.25% trypsin digestion solution was obtained from Beyotime Biotechnology (Shanghai, China); lenvatinib and LY294002 inhibitors were purchased from MedChemExpress(USA); AFP, HK2, LDHA, PI3K, and AKT antibodies were acquired from Abcam (USA); PKM2, P-gp, Bcl-2, and PFK1 antibodies were purchased from Proteintech(USA); and glucose and lactic acid test kits were obtained from Nanjing Jiancheng Bioengineering Institute(Nanjing, China).

Techniques: Western Blot, Expressing, Over Expression

Journal: Discover Oncology

Article Title: AFP confers the resistance of lenvatinib in hepatocellular carcinoma by activating PI3K/AKT/LDHA signaling axis

doi: 10.1007/s12672-026-04637-6

Figure Lengend Snippet: The impact of LDHA on lenvatinib resistance in HCC cells regulated by AFP. a Stable liver cancer cell lines with LDHA overexpression (left) and LDHA knockdown (right) were established. Western blotting assay was performed to verify the stable transfection, the right bar chart represents the statistical analysis of the gray-scale scan values of protein bands. b Cell inhibition rates of HuH-7-shNC, HuH-7-shAFP, HuH-7-shAFP-LDHA cells and HLE-NC, HLE-AFP, HLE-AFP-shLDHA cells after 6 days of treatment with different concentrations of lenvatinib. c Colony formation assay evaluating the clonogenic ability of cells treated with 10 µM lenvatinib. Images were captured at 100 × magnification with a scale bar of 100 µm; "ns" indicates P > 0.05, * P < 0.05, ** P < 0.01. N = 3

Article Snippet: Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum were purchased from Gibco (USA); 0.25% trypsin digestion solution was obtained from Beyotime Biotechnology (Shanghai, China); lenvatinib and LY294002 inhibitors were purchased from MedChemExpress(USA); AFP, HK2, LDHA, PI3K, and AKT antibodies were acquired from Abcam (USA); PKM2, P-gp, Bcl-2, and PFK1 antibodies were purchased from Proteintech(USA); and glucose and lactic acid test kits were obtained from Nanjing Jiancheng Bioengineering Institute(Nanjing, China).

Techniques: Over Expression, Knockdown, Western Blot, Stable Transfection, Inhibition, Colony Assay

Journal: Discover Oncology

Article Title: AFP confers the resistance of lenvatinib in hepatocellular carcinoma by activating PI3K/AKT/LDHA signaling axis

doi: 10.1007/s12672-026-04637-6

Figure Lengend Snippet: The effect of LDHA on AFP-regulated lenvatinib-induced apoptosis in liver cancer cells. a Apoptosis of HuH-7-shNC, HuH-7-shAFP, and HuH-7-shAFP-LDHA cells treated with either no drug or 10 µM lenvatinib; apoptosis of HLE-NC, HLE-AFP, and HLE-AFP-shLDHA cells treated with either no drug or 10 µM lenvatinib, as detected by TUNEL assay. b Cell viability of each group measured by trypan blue staining; white cells indicated by black arrows represent live cells, while blue cells indicated by red arrows represent dead cells. c Expression of drug resistance-rlated protein P-gp and anti-apoptotic protein Bcl-2 were detected by Western blotting, the right bar chart represents the statistical analysis of the gray-scale scan values of protein bands. Magnification: 200 × , scale bar: 50 µm; * P < 0.05, ** P < 0.01. N = 3

Article Snippet: Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum were purchased from Gibco (USA); 0.25% trypsin digestion solution was obtained from Beyotime Biotechnology (Shanghai, China); lenvatinib and LY294002 inhibitors were purchased from MedChemExpress(USA); AFP, HK2, LDHA, PI3K, and AKT antibodies were acquired from Abcam (USA); PKM2, P-gp, Bcl-2, and PFK1 antibodies were purchased from Proteintech(USA); and glucose and lactic acid test kits were obtained from Nanjing Jiancheng Bioengineering Institute(Nanjing, China).

Techniques: TUNEL Assay, Staining, Expressing, Western Blot

Journal: Discover Oncology

Article Title: AFP confers the resistance of lenvatinib in hepatocellular carcinoma by activating PI3K/AKT/LDHA signaling axis

doi: 10.1007/s12672-026-04637-6

Figure Lengend Snippet: The schematic diagram of AFP activating the PI3K/AKT signaling pathway leading to liver cancer cells' tolerance to lenvatinib. AFP activates the PI3K/AKT signaling pathway, stimulating the expression of glucose metabolism reprogramming key enzymes, LDHA, PKM2, HK2 and PFK1 regulated by hypoxia-inducible factor-1α(HIF-1α), and promoting the production of lactic acid in HCC cells. AFP can also promote the expression of P-pg and Bcl-2. Lactic acid, synergizes with P-gp and Bcl-2, inhibits the killing effect of lenvatinib on liver cancer cells

Article Snippet: Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum were purchased from Gibco (USA); 0.25% trypsin digestion solution was obtained from Beyotime Biotechnology (Shanghai, China); lenvatinib and LY294002 inhibitors were purchased from MedChemExpress(USA); AFP, HK2, LDHA, PI3K, and AKT antibodies were acquired from Abcam (USA); PKM2, P-gp, Bcl-2, and PFK1 antibodies were purchased from Proteintech(USA); and glucose and lactic acid test kits were obtained from Nanjing Jiancheng Bioengineering Institute(Nanjing, China).

Techniques: Expressing