Journal: Advanced Science

Article Title: Peptide Transporter 1‐Mediated Dipeptide Transport Promotes Hepatocellular Carcinoma Metastasis by Activating MAP4K4/G3BP2 Signaling Axis

doi: 10.1002/advs.202306671

Figure Lengend Snippet: PEPT1‐mediated HCC metastasis was dependent on MAP4K4. A) Protein expression of EMT‐associated proteins in HCC cells with PEPT1 overexpression or knockdown. B) Volcano plot of all differential genes in Huh7 cells that stably express shRNA stargeting PEPT1 or scramble control. C) Go analysis showed that differentially expressed genes were mainly enriched in protein kinase binding. D) The protein expression of MAP4K4 in PEPT1‐overexpression or PEPT1‐silencing HCC cells was detected by Western blot analysis. E) MAP4K4 protein levels in fresh HCC and adjacent nontumor tissues detection by Western blot ( n = 12). F) Representative IHC images of MAP4K4 in HCC tissue ( n = 10) and corresponding normal tissue ( n = 10). Scale bar, 100 µm. G) The correlation between PEPT1 and MAP4K4 was analyzed based on HCC date from the ICJC (LIRI‐JP) database (left) and Western blot results (right). H) Kaplan–Meier analysis of overall survival (OS) data from ICJC (LIRI‐JP) liver cancer database. I) Representative images and quantification of the indicated cells in the wound‐healing migration assays. Scale bar, 100 µm. J) Representative images and quantification of the migration and invasion of the indicated cells in the Transwell assays. Scale bar, 250 µm. K) Protein expression of EMT‐associated proteins in HCC cells with MAP4K4 knockdown. L) Representative images and quantification of the migration and invasion of the indicated cells in the Transwell assays. Scale bar, 250 µm. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: [ ] Briefly, after the indicated treatments, the cells were cultured in a 15 mm glass‐bottom confocal dish for 24 h. The cells were then washed with phosphate buffered saline (PBS), fixed with 4% paraformaldehyde for 15 min, and permeabilized with 0.5% Triton X‐100 for 20 min. After blocking, the slides were staining overnight at 4 °C with indicated primary antibodies: HA (1:50, Proteintech, #51064‐2‐AP), Flag (1:2000, Proteintech, #66008‐4‐Ig), and MAP4K4 (1:50, Cusabio, #CSB‐PA013439LA01HU).

Techniques: Expressing, Over Expression, Knockdown, Stable Transfection, shRNA, Control, Binding Assay, Western Blot, Migration

Journal: Advanced Science

Article Title: Peptide Transporter 1‐Mediated Dipeptide Transport Promotes Hepatocellular Carcinoma Metastasis by Activating MAP4K4/G3BP2 Signaling Axis

doi: 10.1002/advs.202306671

Figure Lengend Snippet: MAP4K4 directly binds to G3BP2 in HCC. A) Phosphorylated proteomics analysis identified G3BP2 in the binding protein pool. B,C) Endogenous interaction between MAP4K4 and G3BP2 was determined using co‐IP with anti‐MAP4K4 or anti‐G3BP2 antibodies in Huh7 and PLC/PRF/5 cells. D) Exogenous interaction between MAP4K4 and G3BP2 was determined using co‐IP with anti‐Flag or anti‐HA antibodies in HEK 293T cells co‐transfected with Flag‐G3BP2 and HA‐MAP4K4. E) Immunofluorescence staining showing colocalization of endogenous MAP4K4 (red) and G3BP2 (green) in Huh7 and PLC/PRF/5 cells. The nucleus is labeled by DAPI (blue). Scale bar: 20 µm. F) Immunofluorescence staining showing colocalization of exogenous HA‐MAP4K4 (red) and Flag‐G3BP2 (green) in HEK293T cells. The nucleus is labeled by DAPI (blue). Scale bar: 20 µm. G) Representative IHC images for MAP4K4 and G3BP2 in pulmonary metastatic lesions of nude mice developed by PEPT1‐knockdown or PEPT1‐overexpression HCC cells. Scale bar, 500 µm, 50 µm.

Article Snippet: [ ] Briefly, after the indicated treatments, the cells were cultured in a 15 mm glass‐bottom confocal dish for 24 h. The cells were then washed with phosphate buffered saline (PBS), fixed with 4% paraformaldehyde for 15 min, and permeabilized with 0.5% Triton X‐100 for 20 min. After blocking, the slides were staining overnight at 4 °C with indicated primary antibodies: HA (1:50, Proteintech, #51064‐2‐AP), Flag (1:2000, Proteintech, #66008‐4‐Ig), and MAP4K4 (1:50, Cusabio, #CSB‐PA013439LA01HU).

Techniques: Binding Assay, Co-Immunoprecipitation Assay, Transfection, Immunofluorescence, Staining, Labeling, Knockdown, Over Expression

Journal: Advanced Science

Article Title: Peptide Transporter 1‐Mediated Dipeptide Transport Promotes Hepatocellular Carcinoma Metastasis by Activating MAP4K4/G3BP2 Signaling Axis

doi: 10.1002/advs.202306671

Figure Lengend Snippet: MAP4K4‐mediated phosphorylation at T227 is required for the function of G3BP2 in HCC metastasis. A) HEK293T cells were transfected with vectors, Flag‐G3BP2 along with HA‐MAP4K4. The cell extracts were used to immunoprecipitated Flag‐G3BP2 and blotted with anti‐p‐Ser/Thr/Tyr and anti‐Flag antibodies. The whole‐cell lysate (WCL) was blotted with anti‐HA, anti‐Flag, and anti‐β‐actin antibodies. B) The MAP4K4 knockdown Huh7 and PLC/PRF/5 extracts were used to immunoprecipitated G3BP2 and blotted with anti‐p‐Ser/Thr/Tyr and anti‐G3BP2 antibodies. The WCL was blotted with anti‐MAP4K4, anti‐G3BP2, and anti‐β‐actin antibodies. C) Schematic diagram of G3BP2 structure and phosphorylation sites. D) HEK293T cells were transfected with Flag‐G3BP2 (WT), Flag‐G3BP2 (T227A), or HA‐MAP4K4 as the indicated combinations. The cell extracts were used to immunoprecipitated Flag‐G3BP2 and blotted with anti‐p‐Ser/Thr/Tyr and anti‐Flag antibodies. The WCL was blotted with anti‐HA, anti‐Flag and anti‐β‐actin antibodies. E) Huh7 and PLC/PRF/5 cells were transfected with vector, G3BP2 (WT) or G3BP2 (T227A), and the protein expression of MAP4K4, G3BP2, EMT‐associated proteins were detected by Western blot. F) Representative images and quantification of the migration and invasion of the indicated cells in the Transwell assays. Scale bar, 250 µm. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: [ ] Briefly, after the indicated treatments, the cells were cultured in a 15 mm glass‐bottom confocal dish for 24 h. The cells were then washed with phosphate buffered saline (PBS), fixed with 4% paraformaldehyde for 15 min, and permeabilized with 0.5% Triton X‐100 for 20 min. After blocking, the slides were staining overnight at 4 °C with indicated primary antibodies: HA (1:50, Proteintech, #51064‐2‐AP), Flag (1:2000, Proteintech, #66008‐4‐Ig), and MAP4K4 (1:50, Cusabio, #CSB‐PA013439LA01HU).

Techniques: Phospho-proteomics, Transfection, Immunoprecipitation, Knockdown, Plasmid Preparation, Expressing, Western Blot, Migration

Journal: Advanced Science

Article Title: Peptide Transporter 1‐Mediated Dipeptide Transport Promotes Hepatocellular Carcinoma Metastasis by Activating MAP4K4/G3BP2 Signaling Axis

doi: 10.1002/advs.202306671

Figure Lengend Snippet: G3BP2 was upregulated in HCC and influenced cell migration and invasion. A) G3BP2 protein levels in fresh HCC and adjacent nontumor tissues detected by Western blot ( n = 12). B) Representative IHC images of G3BP2 in HCC tissue ( n = 10) and corresponding normal tissue ( n = 10). Scale bar, 100 µm. C) The correlation between PEPT1 and G3BP2 (left), and MAP4K4 and G3BP2 (right) was analyzed based on the Western blot results. D) Representative images and quantification of the indicated cells in the wound‐healing migration assays. Scale bar, 100 µm. E) Representative images and quantification of the migration and invasion of the indicated cells in the Transwell assays. Scale bar, 250 µm. F) Protein expression of EMT‐associated proteins in HCC cells with G3BP2 knockdown. G) Representative images and quantification of the migration and invasion of the indicated cells in the Transwell assays. Scale bar, 250 µm. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: [ ] Briefly, after the indicated treatments, the cells were cultured in a 15 mm glass‐bottom confocal dish for 24 h. The cells were then washed with phosphate buffered saline (PBS), fixed with 4% paraformaldehyde for 15 min, and permeabilized with 0.5% Triton X‐100 for 20 min. After blocking, the slides were staining overnight at 4 °C with indicated primary antibodies: HA (1:50, Proteintech, #51064‐2‐AP), Flag (1:2000, Proteintech, #66008‐4‐Ig), and MAP4K4 (1:50, Cusabio, #CSB‐PA013439LA01HU).

Techniques: Migration, Western Blot, Expressing, Knockdown

Journal: Advanced Science

Article Title: Peptide Transporter 1‐Mediated Dipeptide Transport Promotes Hepatocellular Carcinoma Metastasis by Activating MAP4K4/G3BP2 Signaling Axis

doi: 10.1002/advs.202306671

Figure Lengend Snippet: PEPT1‐mediated dipeptides transport was essential for activating MAP4K4/G3BP2 axis. A) Endogenous interaction between PEPT1 and MAP4K4 was determined using co‐IP with anti‐MAP4K4 antibodies in Huh7 and PLC/PRF/5 cells. B) Metabolomics analysis identified dipeptides downregulated in Huh7 cells with stably PEPT1 knockdown. C) The uptake of Pro‐Gly in HCC cells with PEPT1 overexpressing and knockdown. D) Representative images and quantification of the migration and invasion of the indicated cells in the Transwell assays. Scale bar, 250 µm. E) Huh7 and PLC/PRF/5 cells were incubated with the indicated concentrations of Ile‐Ala or Gln‐Tyr for 24 hours, and the protein expression of MAP4K4, G3BP2, EMT‐associated proteins were detected by Western blot. Error bars indicate means ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: [ ] Briefly, after the indicated treatments, the cells were cultured in a 15 mm glass‐bottom confocal dish for 24 h. The cells were then washed with phosphate buffered saline (PBS), fixed with 4% paraformaldehyde for 15 min, and permeabilized with 0.5% Triton X‐100 for 20 min. After blocking, the slides were staining overnight at 4 °C with indicated primary antibodies: HA (1:50, Proteintech, #51064‐2‐AP), Flag (1:2000, Proteintech, #66008‐4‐Ig), and MAP4K4 (1:50, Cusabio, #CSB‐PA013439LA01HU).

Techniques: Co-Immunoprecipitation Assay, Stable Transfection, Knockdown, Migration, Incubation, Expressing, Western Blot

Journal: Advanced Science

Article Title: Peptide Transporter 1‐Mediated Dipeptide Transport Promotes Hepatocellular Carcinoma Metastasis by Activating MAP4K4/G3BP2 Signaling Axis

doi: 10.1002/advs.202306671

Figure Lengend Snippet: PEPT1/MAP4K4/G3BP2 signaling axis promoted HCC metastasis. A,C) Western blot analysis showed that the knockdown efficacy of MAP4K4 or G3BP2 in Bel7405 and HCCLM3 cells with PEPT1 overexpressing. B,D) Transwell assays revealed that migration and invasion ability was abrogated in Bel7405 and HCCLM3 cells with MAP4K4 or G3BP2 knockdown compared with the control group. Scale bar, 250 µm. E,G) Western blot analysis showed that the overexpression efficacy of G3BP2 (or PEPT1) in Huh7 cells with MAP4K4 (or G3BP2) knockdown. F,H) Inhibited migration and invasion of Huh7 cells with MAP4K4 (or G3BP2) knocked down was rescued by the overexpression of G3BP2 (or PEPT1). Left, representative images of Transwell assays, scale bar, 250 µm. Right, statistical analysis of Transwell assays. I) A schematic diagram of the PEPT1/MAP4K4/G3BP2 regulatory signaling axis that facilitates HCC metastasis.

Article Snippet: [ ] Briefly, after the indicated treatments, the cells were cultured in a 15 mm glass‐bottom confocal dish for 24 h. The cells were then washed with phosphate buffered saline (PBS), fixed with 4% paraformaldehyde for 15 min, and permeabilized with 0.5% Triton X‐100 for 20 min. After blocking, the slides were staining overnight at 4 °C with indicated primary antibodies: HA (1:50, Proteintech, #51064‐2‐AP), Flag (1:2000, Proteintech, #66008‐4‐Ig), and MAP4K4 (1:50, Cusabio, #CSB‐PA013439LA01HU).

Techniques: Western Blot, Knockdown, Migration, Control, Over Expression