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

Journal: Neural Regeneration Research

Article Title: MAP4K4 induces early blood-brain barrier damage in a murine subarachnoid hemorrhage model

doi: 10.4103/1673-5374.290904

Figure Lengend Snippet: Intervention information of experiment II

Article Snippet: A 2 μL volume of recombinant MAP4K4 protein (Abnova, Taiwan, China) solution was infused at a rate of 0.2 μL/min at 1 hour after SAH induction, while 2 μL of MAP4K4 siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 500 pmol in a 2-μL mixture of 1:1 RNase-free water and lipofectamine) or Scr siRNA (Santa Cruz Biotechnology; 500 pmol in a 2-μL mixture of 1:1 RNase-free water and lipofectamine) was infused at an identical rate at 48 hours before SAH induction.

Techniques: Immunofluorescence, Staining

Journal: Neural Regeneration Research

Article Title: MAP4K4 induces early blood-brain barrier damage in a murine subarachnoid hemorrhage model

doi: 10.4103/1673-5374.290904

Figure Lengend Snippet: Intervention information of experiment III

Article Snippet: A 2 μL volume of recombinant MAP4K4 protein (Abnova, Taiwan, China) solution was infused at a rate of 0.2 μL/min at 1 hour after SAH induction, while 2 μL of MAP4K4 siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 500 pmol in a 2-μL mixture of 1:1 RNase-free water and lipofectamine) or Scr siRNA (Santa Cruz Biotechnology; 500 pmol in a 2-μL mixture of 1:1 RNase-free water and lipofectamine) was infused at an identical rate at 48 hours before SAH induction.

Techniques: Western Blot, Zymography

Journal: Neural Regeneration Research

Article Title: MAP4K4 induces early blood-brain barrier damage in a murine subarachnoid hemorrhage model

doi: 10.4103/1673-5374.290904

Figure Lengend Snippet: Time course of intrinsic MAP4K4 expression after SAH (Experiment I). (A) Representative western blot bands of MAP4K4 expression after SAH. (B) Quantitative analysis of MAP4K4 from the ipsilateral hemisphere; the relative density of each protein was normalized to the sham group. Data are expressed as the mean ± SD ( n = 6; one-way analysis of variance and Tukey’s post hoc test). * P < 0.05, vs . sham group. (C) Representative immunohistochemistry of MAP4K4 (red) and lectin (green) in the sham and SAH group at 24 hours after SAH ( n = 3). Scale bar: 20 μm. DAPI: 4′,6-Diamidino-2-phenylindole; MAP4K4: sterile-20-like mitogen-activated protein kinase kinase kinase kinase 4; SAH: subarachnoid hemorrhage.

Article Snippet: A 2 μL volume of recombinant MAP4K4 protein (Abnova, Taiwan, China) solution was infused at a rate of 0.2 μL/min at 1 hour after SAH induction, while 2 μL of MAP4K4 siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 500 pmol in a 2-μL mixture of 1:1 RNase-free water and lipofectamine) or Scr siRNA (Santa Cruz Biotechnology; 500 pmol in a 2-μL mixture of 1:1 RNase-free water and lipofectamine) was infused at an identical rate at 48 hours before SAH induction.

Techniques: Expressing, Western Blot, Immunohistochemistry, Sterility

Journal: Neural Regeneration Research

Article Title: MAP4K4 induces early blood-brain barrier damage in a murine subarachnoid hemorrhage model

doi: 10.4103/1673-5374.290904

Figure Lengend Snippet: Neurological functions, brain water content, and blood-brain barrier permeability after recombinant MAP4K4 delivery and MAP4K4 silencing post SAH (Experiment II). (A) SAH grading scores. (B, C) Representative western blot bands of MAP4K4 siRNA knockdown efficiency at 24 hours post SAH. (D, E) Modified Garcia scores and beam balance test results at 24 and 72 hours. (F, G) Brain water content at 24 and 72 hours after SAH. (H) Evans blue leakage in the ipsilateral hemisphere 24 hours post SAH. Data are expressed as the mean ± SD ( n = 6; one-way analysis of variance and Tukey’s post hoc test). * P < 0.05, vs . sham group; # P < 0.05, vs . SAH + NS group; † P < 0.05, vs . SAH + Scr siRNA group. MAP4K4: Sterile-20-like mitogen-activated protein kinase kinase kinase kinase 4; SAH: subarachnoid hemorrhage; Scr: scramble.

Article Snippet: A 2 μL volume of recombinant MAP4K4 protein (Abnova, Taiwan, China) solution was infused at a rate of 0.2 μL/min at 1 hour after SAH induction, while 2 μL of MAP4K4 siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 500 pmol in a 2-μL mixture of 1:1 RNase-free water and lipofectamine) or Scr siRNA (Santa Cruz Biotechnology; 500 pmol in a 2-μL mixture of 1:1 RNase-free water and lipofectamine) was infused at an identical rate at 48 hours before SAH induction.

Techniques: Permeability, Recombinant, Western Blot, Knockdown, Modification, Sterility

Journal: Neural Regeneration Research

Article Title: MAP4K4 induces early blood-brain barrier damage in a murine subarachnoid hemorrhage model

doi: 10.4103/1673-5374.290904

Figure Lengend Snippet: Continuous endothelial cell and tight junction protein disruption after recombinant MAP4K4 delivery and MAP4K4 silencing post SAH (Experiment III). Endothelium and tight junction discontinuity was demonstrated by lectin and claudin 5 staining in the ipsilateral cortex after MAP4K4 recombinant protein infusion and MAP4K4 siRNA pretreatment, at 24 hours post SAH, under a fluorescence microscope. n = 3. Scale bar: 20 μm. Endothelium and tight junction discontinuity was greater in the SAH group than in the sham group. MAP4K4 increased endothelium and tight junction discontinuity while MAP4K4 siRNA reversed those trends. Arrows indicate discontinuity of endothelial cells and claudin 5. Red: claudin 5; green: lectin; blue: DAPI. DAPI: 4′,6-diamidino-2-phenylindole; MAP4K4: sterile-20-like mitogen-activated protein kinase kinase kinase kinase 4; SAH: subarachnoid hemorrhage; Scr: scramble.

Article Snippet: A 2 μL volume of recombinant MAP4K4 protein (Abnova, Taiwan, China) solution was infused at a rate of 0.2 μL/min at 1 hour after SAH induction, while 2 μL of MAP4K4 siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 500 pmol in a 2-μL mixture of 1:1 RNase-free water and lipofectamine) or Scr siRNA (Santa Cruz Biotechnology; 500 pmol in a 2-μL mixture of 1:1 RNase-free water and lipofectamine) was infused at an identical rate at 48 hours before SAH induction.

Techniques: Disruption, Recombinant, Staining, Fluorescence, Microscopy, Sterility

Journal: Neural Regeneration Research

Article Title: MAP4K4 induces early blood-brain barrier damage in a murine subarachnoid hemorrhage model

doi: 10.4103/1673-5374.290904

Figure Lengend Snippet: Protein expression of nuclear p-p65, tight junction proteins, and MMP9 activity in the ipsilateral hemisphere after recombinant MAP4K4 delivery and MAP4K4 silencing post SAH (Experiment III). (A–D) Protein levels of p-p65, ZO-1, and claudin 5. p-p65 expression levels were elevated at 24 hours, and this effect was enhanced by MAP4K4 infusion and reversed by pretreatment with MAP4K4 siRNA. ZO-1 and claudin 5 expression had the opposite trend. (E, F) Representative gelatin zymography of MMP9 expression revealed that proteinase activity rose at 24 hours after SAH, and was enhanced by MAP4K4 infusion and reversed by pretreatment with MAP4K4 siRNA. The quantified density was normalized to the sham group. Data are expressed as the mean ± SD ( n = 6; one-way analysis of variance and Tukey’s post hoc test). * P < 0.05, vs . sham group; # P < 0.05, vs . SAH + NS group; † P < 0.05, vs . SAH + Scr siRNA group. MAP4K4: Sterile-20-like mitogen-activated protein kinase kinase kinase kinase 4; MMP9: matrix metalloproteinase 9; SAH: subarachnoid hemorrhage; Scr: scramble.

Article Snippet: A 2 μL volume of recombinant MAP4K4 protein (Abnova, Taiwan, China) solution was infused at a rate of 0.2 μL/min at 1 hour after SAH induction, while 2 μL of MAP4K4 siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 500 pmol in a 2-μL mixture of 1:1 RNase-free water and lipofectamine) or Scr siRNA (Santa Cruz Biotechnology; 500 pmol in a 2-μL mixture of 1:1 RNase-free water and lipofectamine) was infused at an identical rate at 48 hours before SAH induction.

Techniques: Expressing, Activity Assay, Recombinant, Zymography, Sterility

Journal: Neural Regeneration Research

Article Title: MAP4K4 induces early blood-brain barrier damage in a murine subarachnoid hemorrhage model

doi: 10.4103/1673-5374.290904

Figure Lengend Snippet: Neurological function, brain water content, and blood-brain barrier leakage after PF delivery post SAH (Experiment IV). (A, B) SAH grading scores. (B, C) Modified Garcia scores and beam balance test results. (D, E) Brain water content at 24 and 72 hours after SAH. (F) Evans blue leakage at 24 hours. Data are expressed as the mean ± SD ( n = 6; one-way analysis of variance and Tukey’s post hoc test). * P < 0.05, vs . sham group; # P < 0.05, vs . SAH + vehicle group. MAP4K4: Sterile-20-like mitogen-activated protein kinase kinase kinase kinase 4; PF: PF-06260933; SAH: subarachnoid hemorrhage.

Article Snippet: A 2 μL volume of recombinant MAP4K4 protein (Abnova, Taiwan, China) solution was infused at a rate of 0.2 μL/min at 1 hour after SAH induction, while 2 μL of MAP4K4 siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 500 pmol in a 2-μL mixture of 1:1 RNase-free water and lipofectamine) or Scr siRNA (Santa Cruz Biotechnology; 500 pmol in a 2-μL mixture of 1:1 RNase-free water and lipofectamine) was infused at an identical rate at 48 hours before SAH induction.

Techniques: Modification, Sterility