rabbit anti map4k4 antibody  (Cell Signaling Technology Inc)

Journal: The Journal of Biological Chemistry

Article Title: Blockade of a novel MAP4K4-LATS2-SASH1-YAP1 cascade inhibits tumorigenesis and metastasis in luminal breast cancer

doi: 10.1016/j.jbc.2024.107309

Figure Lengend Snippet: SASH1 binds to MAP4K4. A , proteins that likely bind to SASH1 in stable SK-BR-3 cells were identified by LC/LC MS analysis. B , the peptide sequences of the immunoprecipitated SASH1 complexes identified by twice LC‒MS/MS and bioinformatics analyses. C , the association between endogenous SASH1 and endogenous MAP4K4 was identified by IP-WB in SK-BR-3 cells. D , the association between exogenous SASH1 and endogenous MAP4K4 in SK-BR-3 cells was identified by IP-WB. E – G , schematic diagram of the SASH1 mutants. The N-terminal domain (aa 1–581) of SASH1 binds to MAP4K4. H , the colocalization of SASH1 and MAP4K4 in SK-BR-3, T47D and MCF-7 cells. Immunofluorescence laser scanning microscopy was used to visualize the expression of endogenous SASH1 and endogenous MAP4K4 in SK-BR-3, T47D, and MCF-7 cells after seeding on coverslips. SK-BR-3, T47D, and MCF-7 cells were subjected to immunofluorescence (IF) staining with an anti-SASH1 antibody ( green ) and an anti-MAP4K4 antibody ( red ) and were evaluated under a confocal microscope. Negative controls in which the primary antibody was not added during IF staining were established. Scale bar: 20 μm.

Article Snippet: The cells were incubated with a mouse anti-SASH1 monoclonal antibody (MA5-24573, Thermo Fisher) and a rabbit anti-MAP4K4 polyclonal antibody (A301-502A, Bethyl Laboratories) overnight at 4 °C.

Techniques: Liquid Chromatography with Mass Spectroscopy, Immunoprecipitation, Immunofluorescence, Laser-Scanning Microscopy, Expressing, Staining, Microscopy

Journal: The Journal of Biological Chemistry

Article Title: Blockade of a novel MAP4K4-LATS2-SASH1-YAP1 cascade inhibits tumorigenesis and metastasis in luminal breast cancer

doi: 10.1016/j.jbc.2024.107309

Figure Lengend Snippet: SASH1 expression is negatively correlated with MAP4K4 expression in luminal-subtype breast cancer tissues. A , a representative image of IHC staining for SASH1 and MAP4K4 in 30 benign breast tissues. Correlation analysis of the SASH1 and MAP4K4 scores revealed that SASH1 expression was positively correlated with MAP4K4 expression ( right panel ). B , correlation analysis of MAP4K4 and SASH1 expression in 111 normal breast specimens suggested that MAP4K4 expression was significantly associated with SASH1 expression at the transcriptional level. The data were obtained from The Cancer Genome Atlas (TCGA) ( https://www.cancer.gov/about-nci/organization/ccg/research/structural-genomics/tcga ). C , representative image of IHC staining for SASH1 and MAP4K4 in 119 luminal-subtype breast cancer tissues, namely, 64 luminal A and 55 luminal B tissues. In the luminal subtype tissues, SASH1 expression was significantly negatively associated with MAP4K4 expression. D , a representative image of IHC staining for SASH1 and MAP4K4 in 17 Her2-positive subtype breast cancer tissues. In the Her2-positive subtype tissues, SASH1 expression was not associated with MAP4K4 expression. E , a representative image of IHC staining for SASH1 and MAP4K4 in 14 TNBC subtype breast cancer tissues. In the TNBC subtype tissues, SASH1 expression was not associated with MAP4K4 expression. Magnification of the images in Figure 2 , A – E : 10× ; scale bar: 10 μm. F , correlation analysis of MAP4K4 and SASH1 expression in 1054 breast cancer specimens from the TCGA database revealed that MAP4K4 expression was not associated with SASH1 expression at the transcriptional level. The extent of the correlation between SASH1 and MAP4K4 was identified via bivariate linear correlation analysis in SPSS v.16.0 software. GraphPad Prism v.6.0 software was used to construct the figures. G , decreased expression of SASH1 and increased expression of MAP4K4 were detected in 10 representative luminal-subtype breast cancer tissues compared to the paired paracancerous tissues. N: paracancerous tissues, C: cancer tissues. H , SASH1 downregulation and MAP4K4 upregulation were detected via western blotting in various breast cancer cell lines compared with normal breast cells (MCF10A).

Article Snippet: The cells were incubated with a mouse anti-SASH1 monoclonal antibody (MA5-24573, Thermo Fisher) and a rabbit anti-MAP4K4 polyclonal antibody (A301-502A, Bethyl Laboratories) overnight at 4 °C.

Techniques: Expressing, Immunohistochemistry, Software, Construct, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Blockade of a novel MAP4K4-LATS2-SASH1-YAP1 cascade inhibits tumorigenesis and metastasis in luminal breast cancer

doi: 10.1016/j.jbc.2024.107309

Figure Lengend Snippet: MAP4K4 upregulation and SASH1 downregulation cooperate to regulate the proliferation, migration and invasion of ER-positive breast cancer cells. A , an increase in the proportion of S-phase cells resulted from SASH1 silencing in T47D cells stably transduced with SASH1-shRNA. Stable T47D cells were starved for 24 h, and the cell cycle distribution of T47D cells was analyzed using flow cytometry. B , SASH1 silencing inhibited the apoptosis of T47D cells. Flow cytometry assays indicated that SASH1 silencing caused a significant increase in the proportion of apoptotic cells. C and D , increases in the numbers of migrating and invading T47D cells resulted from SASH1 silencing. Transwell migration and invasion assays were performed after stable T47D cells were starved for 24 h. The numbers of migrated and invaded cells in the evaluated fields of view were counted and analyzed statistically. E , CCK-8 cell proliferation assays indicated that cell viability was increased by SASH1 silencing in T47D cells. F , SASH1 silencing in T47D cells was evaluated by western blotting. G , an increase in the proportion of S-phase cells resulted from MAP4K4 overexpression and/or SASH1 silencing in T47D cells. T47D cells were infected with MAP4K4 -overexpressing adenovirus and/or SASH1 -shRNA lentivirus, and after 24 h of infection, the cells were starved for approximately 24 h. The cell cycle distribution of the T47D cells was analyzed using flow cytometry. H , a decrease in the proportion of apoptotic cells resulted from MAP4K4 overexpression and/or SASH1 silencing in T47D cells. The proportion of S-phase cells and percentage of apoptotic cells are expressed as the means ± SDs, and one-way ANOVA was used for multiple comparisons in Figure 3 , A , B , G and H . I , increased migration of T47D cells was triggered by MAP4K4 overexpression and/or SASH1 silencing. Transwell migration assays were performed after T47D cells were infected with SASH1 -shRNA lentivirus and/or MAP4K4 overexpression adenovirus and starved for 24 h. The numbers of migrated and invaded T47D cells are expressed as the means ± SDs, and one-way ANOVA was used for multiple comparisons in Figure 3 , C , D and I . Scale bar: 50 μm. J , CCK-8 cell proliferation assays indicated that cell viability was increased by SASH1 silencing and/or MAP4K4 overexpression in T47D cells. Cell viability is expressed as the mean ± SD, and one-way ANOVA was used for multiple comparisons in Figure 3 , E and J . K , Myc- MAP4K4 overexpression and SASH1 silencing were evaluated by western blotting in T47D cells infected with myc- MAP4K4 ADV and/or SASH1 -shRNA lentivirus.

Article Snippet: The cells were incubated with a mouse anti-SASH1 monoclonal antibody (MA5-24573, Thermo Fisher) and a rabbit anti-MAP4K4 polyclonal antibody (A301-502A, Bethyl Laboratories) overnight at 4 °C.

Techniques: Migration, Stable Transfection, Transduction, shRNA, Flow Cytometry, CCK-8 Assay, Western Blot, Over Expression, Infection

Journal: The Journal of Biological Chemistry

Article Title: Blockade of a novel MAP4K4-LATS2-SASH1-YAP1 cascade inhibits tumorigenesis and metastasis in luminal breast cancer

doi: 10.1016/j.jbc.2024.107309

Figure Lengend Snippet: SASH1 is phosphorylated through a novel MAP4K4-LATS1/2 cascade, and SASH1 S914 is necessary for SASH1 phosphorylation. A , exogenous LATS1 bound not only to exogenous MAP4K4 but also to exogenous SASH1. HA- LATS1 ; GFP- MAP4K4 and GFP- SASH1 were transfected into HEK-293T cells in different combinations. Forty-eight hours after transfection, HA-LATS1 was immunoprecipitated, and bound GFP-MAP4K4 and GFP-SASH1 were detected by western blotting. B , exogenous LATS2 associated not only with exogenous MAP4K4 but also with exogenous SASH1. C and D , exogenous MAP4K4 interacted with exogenous LATS2 and exogenous LATS1 in HEK-293T cells. E and F , exogenous MAP4K4 did not bind to exogenous MST1 or exogenous MST2 in HEK-293T cells. G , in the presence of MAP4K4, LATS1, and LATS2, the mobility of ectopically expressed SASH1 was notably retarded, and YAP phosphorylation was induced, suggesting that the MAP4K4 and Hippo core kinases induced the phosphorylation of SASH1 and YAP. HEK-293T cells were transfected with HA- AMOT or Hippo core kinases (MST1, LATS1, LATS2, and YAP1) or with GFP- SASH1 , GFP- MAP4K4 or Hippo core kinases (LATS1, LATS2, and YAP). Transfected cells were lysed, and proteins were separated on a Phos-tag gel. The upward shift in the AMOT band induced by the expression of the Hippo core kinases was employed as the positive control. H , SASH1 phosphorylation was severely impaired by the expression of kinase-dead (KD) mutants of MAP4K4 but not by those of LATS1 . HEK-293T cells were transfected with wild-type LATS1 or LATS1 kinase-dead mutants (K125R and K140R) and with wild-type MAP4K4 or the MAP4K4 KD mutant (K54R). GFP-SASH1 phosphorylation was assessed by Phos-tag gel electrophoresis. I , phos-tag gel electrophoresis indicated that expression of the MAP4K4 KD mutant abolished the phosphorylation of GFP-SASH1 induced by MAP4K4 and/or LATS2. J and K , the sequences in SASH1 containing phosphorylation sites were identified by LC‒MS/MS analysis. @ indicates the serine residues undergoing phosphorylation, including Ser355, Ser359, Ser914 and Ser918. L , the conservation of Ser355, Ser359, Ser914, and Ser918 was analyzed by MEGA-X software. M and N , the changes in the mobility of SASH1 induced by the phosphorylation of Ser355, Ser359, Ser914, and Ser918 were compared using Phos-tag gel electrophoresis in the presence of MAP4K4 and LATS1 or LATS2. A significant downward shift in the SASH1 band was induced by the expression of SASH1-S914A. GFP-tagged MAP4K4 and HA-tagged LATS1/2 were transfected into HEK-293T cells. Forty-eight hours after transfection, the cells were lysed and subjected to western blotting. O and P , the significant upward shift in the SASH1 band was abolished by expression of the SASH1 -S914A or SASH1-S914E mutant in the presence of MAP4K4 and LATS1 or LATS2. The expression of SASH1-S914D (a phosphomimetic mutant) restored the upward shift in the SASH1 band induced by MAP4K4 and LATS1 or LATS2. LE, long exposure; SE, short exposure.

Article Snippet: The cells were incubated with a mouse anti-SASH1 monoclonal antibody (MA5-24573, Thermo Fisher) and a rabbit anti-MAP4K4 polyclonal antibody (A301-502A, Bethyl Laboratories) overnight at 4 °C.

Techniques: Phospho-proteomics, Transfection, Immunoprecipitation, Western Blot, Expressing, Positive Control, Mutagenesis, Nucleic Acid Electrophoresis, Software

Journal: The Journal of Biological Chemistry

Article Title: Blockade of a novel MAP4K4-LATS2-SASH1-YAP1 cascade inhibits tumorigenesis and metastasis in luminal breast cancer

doi: 10.1016/j.jbc.2024.107309

Figure Lengend Snippet: YAP dephosphorylation is induced by SASH1 silencing and/or ectopic MAP4K4 expression, and downregulation of LATS2, SASH1 and YAP1 results from upregulation of MAP4K4. A , a decrease in the p-YAP level resulted from SASH1 silencing in MCF-7 cells. Two SASH1 -shRNA vectors were transduced separately into MCF-7 cells, and 48 h after transfection, the cells were lysed and subjected to western blotting. B , ectopic MAP4K4 expression in MCF-7 cells induced downregulation of LATS1, LATS2, SASH1 and YAP; phosphorylation of LATS1; and dephosphorylation of YAP. GFP- MAP4K4 was transfected into MCF-7 cells, and 48 h after transfection, the cells were lysed and subjected to western blotting. The protein band densities were quantified with Quantity One software and analyzed via one-way ANOVA for multiple comparisons. The data are expressed as the means ± SDs. ∗∗ p < 0.001 versus control and ∗ p < 0.01 versus control. Phospho-YAP and phospho-LATS1 levels were calculated with the following formula: p-YAP or p-LATS1 level = phosphoprotein band density/total protein band density. C , downregulation of YAP1 and dephosphorylation of YAP1 were induced by MAP4K4 overexpression and SASH1 silencing in MCF-7 cells. The protein densitometry data in Figure 5 , C and L are expressed as the means ± SDs. The data were analyzed with one-way ANOVA for multiple comparisons in SPSS 16.0 software and plotted with GraphPad Prism 6.0. ∗∗ p < 0.001 versus control, ∗ p < 0.01 versus control. D , decreased levels of exogenous SASH1 resulted from the introduction of gradually increasing concentrations of exogenous MAP4K4 in HEK-293T cells. HEK-293T cells were transfected with increasing concentrations of exogenous MAP4K4 (GFP-MAP4K4) and low (0.1 μg) or high (1.0 μg) concentrations of exogenous SASH1. 40 h after transfection, the cells were lysed and subjected to immunoblotting. E , decreased expression of endogenous SASH1 was caused by the introduction of gradually increasing concentrations of exogenous MAP4K4 (GFP-MAP4K4) in SK-BR-3 and MCF-7 cells. F , endogenous SASH1 downregulation resulted from ectopic MAP4K4 expression in T47D cells. T47D cells were infected with myc-MAP4K4 adenovirus. Seventy-two hours after infection, the cells were lysed and subjected to western blotting. G , decreased expression of exogenous LATS2 and YAP1 was caused by the introduction of gradually increasing concentrations of exogenous MAP4K4 (GFP-MAP4K4) in HEK-293T cells. H and I , a downward shift in the endogenous SASH1 band resulted from the cosilencing of MAP4K4 and LATS2 but not from the cosilencing of MAP4K4 and LATS1 , as shown by the Phos-tag assay. J , endogenous MAP4K4 bound to endogenous LATS2 but not to LATS1 in MCF-7 cells. Endogenous MAP4K4 in MCF-7 cells was immunoprecipitated, and bound endogenous LATS1/2 and SASH1 were detected by western blotting. K , endogenous SASH1 associated with LATS2 but not with LATS1 in MCF-7 cells. L , downregulation of YAP1 was induced by ectopic expression of LATS2 and/or MAP4K4, and dephosphorylation of YAP1 resulted from ectopic expression of MAP4K4 or the combined effects of ectopic MAP4K4 and LATS2 expression in MCF-7 cells.

Article Snippet: The cells were incubated with a mouse anti-SASH1 monoclonal antibody (MA5-24573, Thermo Fisher) and a rabbit anti-MAP4K4 polyclonal antibody (A301-502A, Bethyl Laboratories) overnight at 4 °C.

Techniques: De-Phosphorylation Assay, Expressing, shRNA, Transfection, Western Blot, Phospho-proteomics, Software, Control, Over Expression, Infection, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: Blockade of a novel MAP4K4-LATS2-SASH1-YAP1 cascade inhibits tumorigenesis and metastasis in luminal breast cancer

doi: 10.1016/j.jbc.2024.107309

Figure Lengend Snippet: Increased nuclear translocation of YAP1/TAZ and upregulation of ER and PGR were observed in response to MAP4K4 upregulation and SASH1 silencing. A , MAP4K4 overexpression and/or SASH1 silencing promoted YAP/TAZ nuclear translocation in MCF-7 cells. MCF-7 cells were transfected with different combinations of Flag- MAP4K4 and SASH1 -specific shRNAs. Forty-eight hours after transfection, the cells were lysed, and the nuclear and cytoplasmic proteins were separately extracted with nuclear and cytoplasmic protein extraction kits according to the manufacturer’s protocol. GAPDH and Lamin A/C were used as the internal controls for cytoplasmic and nuclear proteins, respectively, to assess cross-contamination between the cytosolic and nuclear fractions and quantify the expression of YAP1 and TAZ in the cytoplasm and nucleus. The blots shown are representative of three independent experiments. N: nucleus, C: cytoplasm. B , MAP4K4 overexpression and SASH1 silencing promoted the interaction of YAP1 with TEAD4 in MCF-7 cells. Flag- MAP4K4 , SASH1- specific shRNA, and GFP- TEAD4 were introduced into MCF-7 cells in different combinations. Forty-eight hours after transfection, the cells were lysed and subjected to IP-WB. Endo-YAP1 was immunoprecipitated with an anti-YAP1 antibody, and bound Flag-TEAD4 was detected with an anti-Flag antibody. The binding of Flag-TEAD4 to endo-YAP1 was analyzed by immunoblotting, quantified by densitometry and normalized to the total abundance of endo-YAP1. Representative blots are shown. C , MAP4K4 overexpression and/or SASH1 silencing promoted the interaction between TEAD4 and YAP1 in MCF-7 cells. Flag-TEAD4 was immunoprecipitated with an anti-Flag antibody, and bound endo-YAP1 was detected with an anti-YAP1 antibody. The binding of endo-YAP1 to Flag-TEAD4 was analyzed by immunoblotting, quantified by densitometry and normalized to the total abundance of Flag-TEAD4. D , MAP4K4 overexpression and SASH1 silencing attenuated the association between YAP1 and TEAD1 in MCF-7 cells. Different combinations of FLAG- MAP4K4 , SASH1 -specific shRNA and GFP- TEAD1 were transfected into MCF-7 cells. Endo-YAP1 was immunoprecipitated with an anti-YAP1 antibody, and bound GFP-TEAD1 was detected with an anti-GFP antibody. The binding of GFP-TEAD1 to endo-YAP1 was analyzed through immunoblotting and normalized to the total abundance of endo-YAP1. The protein densitometry data in Figure 6 , A – D are expressed as the means ± SDs. The data were analyzed with one-way ANOVA for multiple comparisons in SPSS 16.0 software and plotted with GraphPad Prism 6.0. E and F , MAP4K4 overexpression and/or SASH1 silencing promoted the expression of YAP1 target genes, as shown by qRT‒PCR and immunoblotting. The Ct values of CTGF and CYR61 are expressed as the means ± SDs and were analyzed with one-way ANOVA for multiple comparisons. G and H , Significant increases in the expression of ERα , ERβ and PGR resulted from ectopic MAP4K4 expression and/or SASH1 silencing in MCF-7 cells, as indicated by qRT‒PCR. Ectopic MAP4K4 expression and/or SASH1 silencing resulted in increased expression of TP53 . The Ct values of ERα , ERβ , PGR and TP53 are expressed as the means ± SDs and were analyzed with one-way ANOVA for multiple comparisons. I , ectopic MAP4K4 and/or SASH1 silencing increased ERα expression in MCF-7 cells, as indicated by immunoblotting. J , the reduction in the p-YAP1 level in the MAP4K4-LATS-SASH1-YAP1 axis induced by PF-06260933 treatment was abolished by LATS2 silencing but not by LATS1 silencing. MCF-7 cells were transfected with either of two LATS1 siRNAs or LATS2 siRNAs. Twenty-four hours after transfection, the cells were treated with 20 μM PF-06260933 for 12 h, lysed and subjected to western blotting. K , downregulation of MAP4K4 and YAP1 and decreased levels of p-LATS2 resulted from PF-06260933 treatment of MCF-7 cells. MCF-7 cells were treated with 20 μM PF-06260933 for 12 h and lysed. The phosphorylation of endogenous MAP4K4 and SASH1 was assessed by the Phos-tag assay. # p < 0.05, ∗ p < 0.01, ∗∗ p < 0.001 versus control. L , downregulation of ERα, CYR61, CTGF, and YAP1 and an increased level of SASH1 resulted from PF-06260933 treatment of MCF-7 cells. The protein densitometry data in Figure 6 , I – K are expressed as the means ± SDs and were analyzed with one-way ANOVA for multiple comparisons. M , schematic diagram showing that YAP1 dephosphorylation and nuclear translocation of YAP1/TAZ are triggered by the combined effects of MAP4K4 upregulation and SASH1 silencing to activate the MAP4K4-LATS2-SASH1-YAP1/TAZ-TEAD4 cascade. N , schematic diagram showing that YAP1 downregulation and a decrease in the nuclear translocation of YAP1/TAZ resulted from MAP4K4 blockade caused by the use of MAP4K4-specific inhibitors or shRNAs to inhibit the activation of the MAP4K4-LATS2-SASH1-YAP1/TAZ-TEAD4 cascade. The schematic diagram was generated by Figdraw ( www.figdraw.com ).

Article Snippet: The cells were incubated with a mouse anti-SASH1 monoclonal antibody (MA5-24573, Thermo Fisher) and a rabbit anti-MAP4K4 polyclonal antibody (A301-502A, Bethyl Laboratories) overnight at 4 °C.

Techniques: Translocation Assay, Over Expression, Transfection, Protein Extraction, Expressing, shRNA, Immunoprecipitation, Binding Assay, Western Blot, Software, Phospho-proteomics, Control, De-Phosphorylation Assay, Activation Assay, Generated

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: Life science alliance

Article Title: MAP4K4 regulates forces at cell-cell and cell-matrix adhesions to promote collective cell migration.

doi: 10.26508/lsa.202302196

Figure Lengend Snippet: Figure 2. MAP4K4 loss of function stabilizes focal adhesions and increases F-actin bundles. (A) Representative confocal images of A431 clusters treated with DMSO or GNE-495 at 1.0 μM stained for zyxin, a mature focal adhesion marker. (B) Number of zyxin- positive focal adhesions on clusters treated with DMSO or GNE-495 at 1.0 μM. At least eight clusters per experiment, from three independent experiments were analyzed. (C) Schematic representation of the different types of stress fibers. (D) Confocal z-scan projection of representative cell protrusion treated with DMSO or GNE-495 at 1.0 μM

Article Snippet: For immunoblotting, the following primary antibodies were used: rabbit polyclonal anti-HGK (MAP4K4) at 1:1,000 (#3485; Cell Signaling Technology), rabbit polyclonal anti-phospho-myosin light chain 2 (Ser19) at 1:1,000 (#3671; Cell Signaling Technology), rabbit polyclonal anti-non-muscle myosin HC II-A at 1:1,000 (#909802; BioLegend), mouse monoclonal anti-actin at 1:10,000 (MAB1501; Millipore [C4]), rabbit polyclonal anti-moesin at 1:1,000 (#3150; Cell Signaling Technology [Q480]).

Techniques: Staining, Marker

Journal: Life science alliance

Article Title: MAP4K4 regulates forces at cell-cell and cell-matrix adhesions to promote collective cell migration.

doi: 10.26508/lsa.202302196

Figure Lengend Snippet: Figure 4. MAP4K4 loss of function increases tension loading at cell–cell junctions. (A) z-scan projection of representative confocal images of p120-catenin stained A431 clusters, showing the differences in the cell–cell junction morphology of clusters treated with DMSO or GNE-495 at 1.0 μM. (B) Crops of the previous images, indicating parallel or perpendicular F-actin organization at the adherens junction. (C, D) Schematic representation of cell–cell junction tortuosity index calculation (D) Cell–cell junction tortuosity index for A431 clusters control (sgNT) or KO for MAP4K4, or treated with DMSO or GNE-495 at 1.0 μM. At least three junctions of five different clusters per experiment, from three independent experiments were analyzed.

Article Snippet: For immunoblotting, the following primary antibodies were used: rabbit polyclonal anti-HGK (MAP4K4) at 1:1,000 (#3485; Cell Signaling Technology), rabbit polyclonal anti-phospho-myosin light chain 2 (Ser19) at 1:1,000 (#3671; Cell Signaling Technology), rabbit polyclonal anti-non-muscle myosin HC II-A at 1:1,000 (#909802; BioLegend), mouse monoclonal anti-actin at 1:10,000 (MAB1501; Millipore [C4]), rabbit polyclonal anti-moesin at 1:1,000 (#3150; Cell Signaling Technology [Q480]).

Techniques: Staining, Control

Journal: Life science alliance

Article Title: MAP4K4 regulates forces at cell-cell and cell-matrix adhesions to promote collective cell migration.

doi: 10.26508/lsa.202302196

Figure Lengend Snippet: Figure 6. MAP4K4 accumulates in the interface of detaching cells and induces cell scattering. (A) Confocal images acquired during time-lapse of A431 cells expressing eGFP–MAP4K4 WT and E-cadherin–mRuby. Arrow indicates accumulation of eGFP–MAP4K4 at cell–cell contacts during cell detachment. Red squares indicate region that was cropped for timepoints 12, 16, and 20 min. Images were acquired every 30 s, during 30 min. (B) Confocal images of DIC and eGFP, acquired during time-lapse of A431 control cells, or A431 cells expressing eGFP–MAP4K4 WT. Top panel represent control cells, middle

Article Snippet: For immunoblotting, the following primary antibodies were used: rabbit polyclonal anti-HGK (MAP4K4) at 1:1,000 (#3485; Cell Signaling Technology), rabbit polyclonal anti-phospho-myosin light chain 2 (Ser19) at 1:1,000 (#3671; Cell Signaling Technology), rabbit polyclonal anti-non-muscle myosin HC II-A at 1:1,000 (#909802; BioLegend), mouse monoclonal anti-actin at 1:10,000 (MAB1501; Millipore [C4]), rabbit polyclonal anti-moesin at 1:1,000 (#3150; Cell Signaling Technology [Q480]).

Techniques: Expressing, Control