Journal: International Journal of Biological Sciences

Article Title: N 6 -methyladenosine Reader IGF2BP2-modified HMMR Promotes Non-small Cell Lung Cancer Metastasis via Interaction with MAP4K4

doi: 10.7150/ijbs.104097

Figure Lengend Snippet: MAP4K4 inhibitor GNE-495 reduces NSCLC cell metastasis in vivo . (A) Flowchart illustrating the NSCLC cell in vivo metastasis model. (B) Relative mRNA and protein expression levels of HMMR and MMP1 in the A549 cells used to establish the in vivo metastasis model. (C) Photographs showing lung metastatic nodules in mice following injection of A549 cells overexpressing HMMR or the control vector, with treatment by DMSO or GNE-495. Metastatic nodules are indicated by red arrowheads. (D) Quantification of lung metastatic nodules across the four experimental groups. (E) The lung weights of the mice in each group after execution. (F) Hematoxylin and eosin (H&E) staining of lung tissue to assess micrometastatic foci. Representative histological images of micrometastatic foci in the four groups. Micrometastatic foci are indicated by blue arrowheads.

Article Snippet: From the fifth week post-injection, mice received daily intraperitoneal injections of either DMSO (3 mg/kg) or the MAP4K4 inhibitor GNE-495 (HY-100343, MCE, China) (3 mg/kg).

Techniques: In Vivo, Expressing, Injection, Control, Plasmid Preparation, Staining

Journal: Biomedicines

Article Title: Ultraviolet B Exposure Does Not Influence the Expression of YAP mRNA in Human Epidermal Keratinocytes—Preliminary Study

doi: 10.3390/biomedicines13030596

Figure Lengend Snippet: TaqMan ® probes used for PCR.

Article Snippet: MAP4K4 , Hs01101394_m1.

Techniques:

Journal: Biomedicines

Article Title: Ultraviolet B Exposure Does Not Influence the Expression of YAP mRNA in Human Epidermal Keratinocytes—Preliminary Study

doi: 10.3390/biomedicines13030596

Figure Lengend Snippet: Fold change of YAP and its regulators in A431 cell line. Data presented as fold change with geometric standard deviations. Data presented as fold change (geometric mean) with geometric standard deviations.

Article Snippet: MAP4K4 , Hs01101394_m1.

Techniques:

Journal: Biomedicines

Article Title: Ultraviolet B Exposure Does Not Influence the Expression of YAP mRNA in Human Epidermal Keratinocytes—Preliminary Study

doi: 10.3390/biomedicines13030596

Figure Lengend Snippet: Detailed mRNA expression of studied Hippo pathway regulators in primary epidermal keratinocytes under the influence of UVB. Data presented as fold change (geometric mean) with geometric standard deviations.

Article Snippet: MAP4K4 , Hs01101394_m1.

Techniques: Expressing

Journal: Biomedicines

Article Title: Ultraviolet B Exposure Does Not Influence the Expression of YAP mRNA in Human Epidermal Keratinocytes—Preliminary Study

doi: 10.3390/biomedicines13030596

Figure Lengend Snippet: Detailed mRNA expression of studied Hippo pathway regulators in primary epidermal keratinocytes under the influence of narrowband UVB. Data presented as fold change (geometric mean) with geometric standard deviations.

Article Snippet: MAP4K4 , Hs01101394_m1.

Techniques: Expressing

Journal: Biomedicines

Article Title: Ultraviolet B Exposure Does Not Influence the Expression of YAP mRNA in Human Epidermal Keratinocytes—Preliminary Study

doi: 10.3390/biomedicines13030596

Figure Lengend Snippet: mRNA expression of studied Hippo pathway regulators: LATS1 ( A ), LATS2 ( B ), JNK1 ( C ), JNK2 ( D ), MAP4K4 ( E ), ABL1 ( F ) in primary epidermal keratinocytes (3 repetitions) with preserved (dark grey) and silenced (tilted) expression of YAP under the influence of increasing doses of UVB. Data presented as fold change (columns) with geometric standard deviations (bars). Asterisks above columns indicate level of statistical significance (ns—not significant, * p < 0.05, ** p < 0.001) in comparison to controls.

Article Snippet: MAP4K4 , Hs01101394_m1.

Techniques: Expressing, Comparison

Journal: Biomedicines

Article Title: Ultraviolet B Exposure Does Not Influence the Expression of YAP mRNA in Human Epidermal Keratinocytes—Preliminary Study

doi: 10.3390/biomedicines13030596

Figure Lengend Snippet: mRNA expression of studied Hippo pathway regulators: LATS1 ( A ), LATS2 ( B ), JNK1 ( C ), JNK2 ( D ), MAP4K4 ( E ), ABL1 ( F ) in primary epidermal keratinocytes (2 repetitions) with preserved (dark grey) and silenced (tilted) expression of YAP under the influence of increasing doses of narrowband UVB. Data presented as fold change (columns) with geometric standard deviations (bars). Asterisks above columns indicate level of statistical significance (ns—not significant, * p < 0.05, ** p < 0.001) in comparison to controls.

Article Snippet: MAP4K4 , Hs01101394_m1.

Techniques: Expressing, Comparison

Journal: Nature cell biology

Article Title: Heat stress activates YAP/TAZ to induce the heat shock transcriptome

doi: 10.1038/s41556-020-00602-9

Figure Lengend Snippet: (a) HEK293A cells were cultured under high density (upper panel) or in the absence of serum (lower panel), and were subjected to heat shock for the indicated times. YAP phosphorylation was detected by the phos-tag gel. (b) Heat shock does not affect MST1 phosphorylation. HEK293A cells were transiently transfected with GST-MST1. 24 h after transfection, cells were subjected to heat shock for the indicated times. Glutathione Sepharose 4B beads (GE Healthcare) were used to purify GTS-MST1. Phosphorylation of the purified GST-MST1 was analyzed by Western blot with pMST1 (Thr183) antibody. (c) YAP dephosphorylation time course in MST1-rescued or MAP4K4-rescued MM8KO cells upon heat shock. Plasmids for HA-YAP and FLAG-MST1 or FLAG-MAP4K4 were transiently co-transfected into HEK293A MM8 KO cells. 24 h after transfection, cells were subcultured to new plate and reached a medium confluence the next day, treated with serum starvation for 2 h, then subjected to heat shock for indicated durations. YAP phosphorylation was detected by the phos-tag gel. (d) Heat shock does not affect the LATS1-MOB1 interaction. HEK293A cells were transiently co-transfected with FLAG-LATS1 and 3×HA-MOB1. 24 h after transfection, cells were subjected to heat shock for the indicated times. FLAG antibodies were used for immunoprecipitation and the co-precipitated proteins were detected by Western blot. The uppermost panel were normalized against FLAG-LATS1 protein levels. Immunoblotting in panels a-d has been performed two times with similar results. Source data are available online.

Article Snippet: FLAG (M2) (F1804, 1:2,000), FLAG-HRP (M2) (A8592, 1:5,000) and GST (2H3-D10) (SAB4200237, 1:4,000) were purchased from Sigma, MAP4K4 (A301-502A, 1:2,000) and MAP4K7 (A310-985A, 1:1,000) were purchased from Bethyl Laboratories, HSP90α (610418, 1:2,000) was purchased from BD Biosciences, HSP70 (10995-1-AP, 1:4,000), ITCH (20920-1-AP, 1:2,000), SIAH2 (12651-1-AP, 1:2,000) and AKT (10176-2-AP, 1:4,000) was purchased from Proteintech, SRC (JF0947) (ET1702-03, 1:5,000) was purchased from HuaAn Biotechnology Co., Ltd and HSP25 (ADI-SPA-801-D, 1:4,000) was purchased from Enzo Life Sciences.

Techniques: Cell Culture, Phospho-proteomics, Transfection, Purification, Western Blot, De-Phosphorylation Assay, Immunoprecipitation

Journal: Nature cell biology

Article Title: Heat stress activates YAP/TAZ to induce the heat shock transcriptome

doi: 10.1038/s41556-020-00602-9

Figure Lengend Snippet: (a) Deletion of MAP4Ks, but not MST, slightly delays heat shock-induced YAP dephosphorylation. HEK393A WT, MST1/2 DKO, and MAP4K4/6/7 TKO cells under medial confluence were pretreated with 2-DG (left panels) or sorbitol (right panels) and then subjected to heat shock. The phosphorylation of YAP and LATS1 were analyzed by Western blot. (b) MAP4K4/6/7 knockout delays YAP nuclear localization. Cells were stained for YAP/TAZ for immunofluorescent microscopy. Representative pictures from three independent samples are shown. Scale bars,10 μm. (c) Quantification of YAP/TAZ nuclear and cytosolic localization. Data are mean ± s.d.; n = 3 biologically independent samples. Two-way ANOVA test. (d) MST1/2 knockout delays recovery after heat shock. HEK293A WT, MST1/2 DKO, and MAP4K4/6/7 TKO cells were pretreated with 2-DG, then subjected to heat shock for 1 h followed by recovery at 37°C for the indicated durations. (e) Heat shock does not affect MST1 and MAP4K4 kinase activity. Endogenous MST1 (left panel) or MAP4K4 (right panel) immunoprecipitated from heat shocked HEK293A cells was assayed using GST-LATS2 as a substrate. LATS2 phosphorylation was determined with pLATS (Thr1079) antibody. (f) Heat shock increases LATS and MAP4K4 interaction. HEK293A cells were co-transfected with FALG-MAP4K4 and HA-LATS1. 24 h after transfection, cells were subjected to heat shock. HA (left panel) and FLAG (right panel) antibodies were used for immunoprecipitation and the co-precipitated proteins were detected by Western blot. (g) Heat shock increases LATS and MST1 interaction. Experiments were similar to panel f except cells were co-transfected with FLAG-MST1 and HA-LATS1 (left panel) or 3×HA-LATS2 (right panel). (h) YAP re-phosphorylation time course in MST1-rescued or MAP4K4-rescued MM8KO cells after shifting back to 37°C. Plasmids for HA-YAP and FLAG-MST1 or FLAG-MAP4K4 were co-transfected into HEK293A MM8KO cells. One day after transfection, cells were subcultured to new plates and reached medial confluence the next day, serum starved for 2 h, then subjected to heat shock at 43°C for 1 h followed by recovery at 37°C for the indicated durations. Immunoblotting in panels a and d-h has been performed two times with similar results. Source data are available online.

Article Snippet: FLAG (M2) (F1804, 1:2,000), FLAG-HRP (M2) (A8592, 1:5,000) and GST (2H3-D10) (SAB4200237, 1:4,000) were purchased from Sigma, MAP4K4 (A301-502A, 1:2,000) and MAP4K7 (A310-985A, 1:1,000) were purchased from Bethyl Laboratories, HSP90α (610418, 1:2,000) was purchased from BD Biosciences, HSP70 (10995-1-AP, 1:4,000), ITCH (20920-1-AP, 1:2,000), SIAH2 (12651-1-AP, 1:2,000) and AKT (10176-2-AP, 1:4,000) was purchased from Proteintech, SRC (JF0947) (ET1702-03, 1:5,000) was purchased from HuaAn Biotechnology Co., Ltd and HSP25 (ADI-SPA-801-D, 1:4,000) was purchased from Enzo Life Sciences.

Techniques: De-Phosphorylation Assay, Phospho-proteomics, Western Blot, Knock-Out, Staining, Microscopy, Activity Assay, Immunoprecipitation, Transfection

Journal: Nature cell biology

Article Title: Heat stress activates YAP/TAZ to induce the heat shock transcriptome

doi: 10.1038/s41556-020-00602-9

Figure Lengend Snippet: (a) Heat shock-induced YAP dephosphorylation is not altered in HSF-1 KO cells. HEK293A WT and HSF-1 KO cells (generated by CRISPR) were subjected to heat shock and cell lysates were analyzed by Western blot. Two independent HSF-1 KO clones are shown. (b) HSP70-1/2 deletion moderately delays YAP dephosphorylation induced by heat shock. HEK293A WT and HSP70-1/2 DKO cells were subjected to heat shock and phos-tag gel was used to detect YAP phosphorylation. Two independent HSP70 KO clones are shown. (c) Deletion of HSP90α/β compromises dephosphorylation of LATS1 and YAP by heat shock. HEK293A WT and HSP90α/β DKO cells were subjected to heat shock and cell lysates were analysed by Western blot. Two independent HSP90α/β DKO clones are shown. LE denotes long exposure of the Western blot. (d) Knockdown of HSP90α/β compromises the heat shock-induced dephosphorylation of YAP and LATS1. HEK293A cells were transfected with control siRNA or siRNAs for HSP90α and HSP90β. Two independent siRNAs were used. (e) The knockdown efficiency of HSP90α/β was confirmed by quantitative Real-Time PCR. Data are presented as mean ± s.d.; n = 3 biologically independent samples. Two-way ANOVA test. (f) HSP90α/β knockdown delays the heat shock-induced LATS inactivation. Endogenous LATS1 was immunoprecipitated from heat shocked-high density HEK293A cells with HSP90α/β knockdown or overexpression. In vitro kinase assays were performed using recombinant GST-YAP as the substrate. Phosphorylation of GST-YAP was determined by immunoblotting with the pYAP (Ser127) antibody. (g) Heat shock increases the interaction between HSP90α and LATS1 or MAP4K4. The plasmids for FLAG-MAP4K4, HA-LATS1, MYC-HSP90AA1 (left panel) or MYC-HSP90AB1 (right panel) were transiently co-transfected into HEK293A cells. One day after transfection, cells were subjected to heat shock for the indicated times. MYC antibodies were used for immunoprecipition and the associated FLAG-MAP4K4 and HA-LATS1 were detected by Western blot. Immunoblotting in panels a-d, f and g has been performed two times with similar results. Source data are available online.

Article Snippet: FLAG (M2) (F1804, 1:2,000), FLAG-HRP (M2) (A8592, 1:5,000) and GST (2H3-D10) (SAB4200237, 1:4,000) were purchased from Sigma, MAP4K4 (A301-502A, 1:2,000) and MAP4K7 (A310-985A, 1:1,000) were purchased from Bethyl Laboratories, HSP90α (610418, 1:2,000) was purchased from BD Biosciences, HSP70 (10995-1-AP, 1:4,000), ITCH (20920-1-AP, 1:2,000), SIAH2 (12651-1-AP, 1:2,000) and AKT (10176-2-AP, 1:4,000) was purchased from Proteintech, SRC (JF0947) (ET1702-03, 1:5,000) was purchased from HuaAn Biotechnology Co., Ltd and HSP25 (ADI-SPA-801-D, 1:4,000) was purchased from Enzo Life Sciences.

Techniques: De-Phosphorylation Assay, Generated, CRISPR, Western Blot, Clone Assay, Phospho-proteomics, Knockdown, Transfection, Control, Real-time Polymerase Chain Reaction, Immunoprecipitation, Over Expression, In Vitro, Recombinant

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: (A) Representative immunoblotting of MAP4K4 and actin using lysates of A431 control cells (sgNT), or A431 cells KO for MAP4K4 with two independent sgRNA (M4K4_sg1, M4K4_sg2). (B) Mean velocity of A431 clusters control or KO for MAP4K4 , tracked over 5 h of migration. (C) Mean velocity of A431 clusters treated with DMSO or GNE-495 at different doses (0.1, 0.5, or 1.0 μM), over 5 h of treatment. Number of clusters analyzed (sgNT: 34, M4K4_sg1: 48, M4K4_sg2: 35, DMSO: 22, GNE 0.1 μM: 34, GNE 0.5 μM: 26, GNE 1.0 μM: 33), from three independent experiments. (D, E) z-scan projection of representative confocal images of F-actin stained A431 clusters, showing the differences in the actin cytoskeleton organization and in the morphology of clusters control (sgNT) or KO for MAP4K4 (M4K4_sg2) or (E) clusters treated with DMSO or GNE-495 at 1.0 μM for 24 h. Arrows represent the actin arches at protrusion bases and arrowheads indicate retraction fibers. (F) Protrusion area of control/ MAP4K4 KO cells or DMSO/GNE-495–treated cells with indicated doses. At least five clusters per experiment, three protrusions per cluster from three independent experiments were analyzed. (G) Circularity of control/ MAP4K4 KO cell clusters, or clusters treated with DMSO or GNE-495 at indicated doses. At least 25 clusters from three independent experiments were analyzed. (H, I) Mean velocity extension (H) or retraction (I) events at the periphery of the clusters before or after treatment with DMSO or GNE-495 at 1.0 μM, over 5 h of treatment. Number of clusters analyzed (DMSO: 28, GNE 0.1 μM: 26, GNE 0.5 μM: 26, GNE 1.0 μM: 28) from three independent experiments. All the data are presented as mean ± s.d. and tested by Kruskal–Wallis (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: MAP4K4 KO cells were generated using the pLenti.Cas9-blast (#52962; Addgene) construct and the following sgRNA constructs: MAP4K4_sg1 (#76263; Addgene) and MAP4K4_sg2 (#76264; Addgene).

Techniques: Western Blot, Control, Migration, Staining

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: (A) Representative confocal images of pERM staining, at the substrate z focal plane, from clusters treated with DMSO or GNE-495 at 1.0 μM. (B) Quantification of mean intensity of pERM from clusters treated with DMSO or GNE-495 at 1.0 μM. At least 25 clusters from three independent experiments were analyzed. (C, D) Number and (D) length of retraction fibers of clusters treated with DMSO or GNE-495 at 1.0 μM. At least 25 clusters from three independent experiments were analyzed. (E) Representative confocal images of pERM staining at the cell–cell junction z focal plane, from clusters treated with DMSO or GNE-495 at 1.0 μM. (F) Quantification of mean intensity of pERM of clusters treated with DMSO or GNE-495 at 1.0 μM. At least 25 clusters from three independent experiments were analyzed. (G) Representative immunoblotting of moesin and actin from lysates of A431 cells control (Rosa_sg) or KO for MSN using two independent sgRNA sequences ( MSN _sg1, MSN _sg2). (H) Representative confocal images of control or MSN KO clusters stained for zyxin. (I) Number of zyxin-positive focal adhesions in control or MSN KO clusters. (J) Representative confocal z-scan projection of p120 stained cells, control or KO for MSN , showing cell–cell junction morphology. (K) Tortuosity index of cell–cell junction on control or MSN KO cells. At least three junctions of five different clusters per experiment, from three independent experiments were analyzed. All the data are presented as mean ± s.d. and tested by Mann–Whitney test (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: MAP4K4 KO cells were generated using the pLenti.Cas9-blast (#52962; Addgene) construct and the following sgRNA constructs: MAP4K4_sg1 (#76263; Addgene) and MAP4K4_sg2 (#76264; Addgene).

Techniques: Staining, Western Blot, Control, MANN-WHITNEY

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: (A, B, C) z-scan projection of representative confocal images of β-catenin stained A431 clusters, stably expressing (A) eGFP–MAP4K4_WT, (B) eGFP–MAP4K4 kinase dead (MAP4K4 D153N ), or (C) deleted for the CNH domain (MAP4K4 ΔCNH ). Line scan indicates the colocalization between β-catenin and MAP4K4. (D) Immunobloting of MAP4K4 or actin for lysates of A431 cells controls (non-infected or sgNT), KO for MAP4K4 (sg_1 or sg_2) alone or expressing eGFP–MAP4K4 WT, KD, or ΔCNH, resistant to sg_2. (E) Number of zyxin-positive focal adhesions for A431 clusters control (sgNT) or KO for MAP4K4 (M4K4_sg2) and stably expressing eGFP–MAP4K4 WT, KD, or ΔCNH, resistant to sg_2. (F) Cell–cell junction tortuosity index for A431 clusters control (sgNT) or KO for MAP4K4 (M4K4_sg2), and stably expressing eGFP–MAP4K4 WT, KD, or ΔCNH, resistant to sg_2. Data on (E, F) are represented as mean ± s.d. and tested by Kruskal–Wallis (ns, nonsignificant; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: MAP4K4 KO cells were generated using the pLenti.Cas9-blast (#52962; Addgene) construct and the following sgRNA constructs: MAP4K4_sg1 (#76263; Addgene) and MAP4K4_sg2 (#76264; Addgene).

Techniques: Staining, Stable Transfection, Expressing, Western Blot, Infection, Control

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: Membranes were blocked with 5% non‐fat milk (Bio‐Rad) for 1 h at room temperature and then incubated overnight at 4 °C with the following primary antibodies: PEPT1 (1:1000, Huabio, #HA500444), MAP4K4 (1:1000, Cusabio, #CSB‐PA013439LA01HU), G3BP2 (1:1000, Proteintech, #16276‐1‐AP), phospho‐Threonine (1:1000, CST, #9386), E‐cadherin (1:1000, Proteintech, #20874‐1‐AP), N‐cadherin (1:1000, Proteintech, #22018‐1‐AP), vimentin (1:2500, Proteintech, #10366‐1‐AP), β‐catenin (1:1000, Huabio, #HA500444), and β‐actin (1:10 000, Proteintech, #66009‐1‐Ig).

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: Membranes were blocked with 5% non‐fat milk (Bio‐Rad) for 1 h at room temperature and then incubated overnight at 4 °C with the following primary antibodies: PEPT1 (1:1000, Huabio, #HA500444), MAP4K4 (1:1000, Cusabio, #CSB‐PA013439LA01HU), G3BP2 (1:1000, Proteintech, #16276‐1‐AP), phospho‐Threonine (1:1000, CST, #9386), E‐cadherin (1:1000, Proteintech, #20874‐1‐AP), N‐cadherin (1:1000, Proteintech, #22018‐1‐AP), vimentin (1:2500, Proteintech, #10366‐1‐AP), β‐catenin (1:1000, Huabio, #HA500444), and β‐actin (1:10 000, Proteintech, #66009‐1‐Ig).

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: Membranes were blocked with 5% non‐fat milk (Bio‐Rad) for 1 h at room temperature and then incubated overnight at 4 °C with the following primary antibodies: PEPT1 (1:1000, Huabio, #HA500444), MAP4K4 (1:1000, Cusabio, #CSB‐PA013439LA01HU), G3BP2 (1:1000, Proteintech, #16276‐1‐AP), phospho‐Threonine (1:1000, CST, #9386), E‐cadherin (1:1000, Proteintech, #20874‐1‐AP), N‐cadherin (1:1000, Proteintech, #22018‐1‐AP), vimentin (1:2500, Proteintech, #10366‐1‐AP), β‐catenin (1:1000, Huabio, #HA500444), and β‐actin (1:10 000, Proteintech, #66009‐1‐Ig).

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: Membranes were blocked with 5% non‐fat milk (Bio‐Rad) for 1 h at room temperature and then incubated overnight at 4 °C with the following primary antibodies: PEPT1 (1:1000, Huabio, #HA500444), MAP4K4 (1:1000, Cusabio, #CSB‐PA013439LA01HU), G3BP2 (1:1000, Proteintech, #16276‐1‐AP), phospho‐Threonine (1:1000, CST, #9386), E‐cadherin (1:1000, Proteintech, #20874‐1‐AP), N‐cadherin (1:1000, Proteintech, #22018‐1‐AP), vimentin (1:2500, Proteintech, #10366‐1‐AP), β‐catenin (1:1000, Huabio, #HA500444), and β‐actin (1:10 000, Proteintech, #66009‐1‐Ig).

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: Membranes were blocked with 5% non‐fat milk (Bio‐Rad) for 1 h at room temperature and then incubated overnight at 4 °C with the following primary antibodies: PEPT1 (1:1000, Huabio, #HA500444), MAP4K4 (1:1000, Cusabio, #CSB‐PA013439LA01HU), G3BP2 (1:1000, Proteintech, #16276‐1‐AP), phospho‐Threonine (1:1000, CST, #9386), E‐cadherin (1:1000, Proteintech, #20874‐1‐AP), N‐cadherin (1:1000, Proteintech, #22018‐1‐AP), vimentin (1:2500, Proteintech, #10366‐1‐AP), β‐catenin (1:1000, Huabio, #HA500444), and β‐actin (1:10 000, Proteintech, #66009‐1‐Ig).

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: Membranes were blocked with 5% non‐fat milk (Bio‐Rad) for 1 h at room temperature and then incubated overnight at 4 °C with the following primary antibodies: PEPT1 (1:1000, Huabio, #HA500444), MAP4K4 (1:1000, Cusabio, #CSB‐PA013439LA01HU), G3BP2 (1:1000, Proteintech, #16276‐1‐AP), phospho‐Threonine (1:1000, CST, #9386), E‐cadherin (1:1000, Proteintech, #20874‐1‐AP), N‐cadherin (1:1000, Proteintech, #22018‐1‐AP), vimentin (1:2500, Proteintech, #10366‐1‐AP), β‐catenin (1:1000, Huabio, #HA500444), and β‐actin (1:10 000, Proteintech, #66009‐1‐Ig).

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

Journal: Molecular Oncology

Article Title: MAP 4K4 is a novel MAPK / ERK pathway regulator required for lung adenocarcinoma maintenance

doi: 10.1002/1878-0261.12055

Figure Lengend Snippet: MAPK / ERK 1/2 is a downstream signaling mediator of MAP4K4 in lung adenocarcinoma cells. (A) The whole‐cell lysates of different lung adenocarcinoma cell lines, including two KRAS ‐mutant cell lines, A549 and H23; one KRAS and EGFR wild‐type cell line, H1793; three EGFR ‐mutant cell lines, H1650, H1975, and H3255; and one lung bronchus cell line, BEAS ‐2B, were used for IB with indicated antibodies. (B) MAP 4K4‐knockdown cell lines (sh‐M 1 and sh‐M 2) or sh RNA control cell lines (sh‐C) were generated with two different lentiviral‐based sh RNA targeting MAP 4K4 or scrambled sh RNA in H23, H1975, and H1650 cell lines. The whole‐cell lysates were used for IB with indicated antibodies. To detect GTP ‐bound RAS , the cell lysates were incubated with RAF ‐1 RBD agarose. The bound proteins were then resolved by SDS / PAGE and blotted with anti‐ RAS antibody. (C) MAP 4K4‐overexpressing cell lines ( HA ‐M) and control cell lines ( HA ‐C) were established by transfecting pc DNA 3.1‐ HA ‐ MAP 4K4 or pc DNA 3.1‐ HA into A549 or H3255 cell lines followed by G418 selection. The whole‐cell lysates were prepared for IB or subjected to RAS activation assay. (D–F) Constitutively active ERK 2 (act ERK 2) or vector was transfected into MAP 4K4‐knockdown cell lines (sh‐M 1) with Polyjet In Vitro DNA Transfection Reagent. Data in column charts were shown as means ± SD ; ** and # denote a statistically significant difference ( P < 0.01) and no statistically significant difference ( P > 0.05), respectively, compared with sh RNA control cell lines (sh‐C). (D) Left panel: representative pictures of soft agar assay. Right panel: quantification of soft agar assay. (E) Left panel: representative pictures of in vitro cell invasion assay. Right panel: quantification of in vitro cell invasion assay. (F) The whole‐cell lysates of different cell lines were used for IB with indicated antibodies. (G) H1975‐sh‐control (sh‐C) and H1975‐sh‐ MAP 4K4 (sh‐M 1 and sh‐M 2) cells were treated with 3 μ m of erlotinib for 6 and 24 h. IB was performed with indicated antibodies.

Article Snippet: The sections were incubated overnight with MAP4K4 antibody (Biorbyt, San Francisco, CA, USA) in a humidified chamber at room temperature.

Techniques: Mutagenesis, Knockdown, Control, Generated, Incubation, SDS Page, Selection, Activation Assay, Plasmid Preparation, Transfection, In Vitro, Soft Agar Assay, Invasion Assay

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: (A) Representative immunoblotting of MAP4K4 and actin using lysates of A431 control cells (sgNT), or A431 cells KO for MAP4K4 with two independent sgRNA (M4K4_sg1, M4K4_sg2). (B) Mean velocity of A431 clusters control or KO for MAP4K4 , tracked over 5 h of migration. (C) Mean velocity of A431 clusters treated with DMSO or GNE-495 at different doses (0.1, 0.5, or 1.0 μM), over 5 h of treatment. Number of clusters analyzed (sgNT: 34, M4K4_sg1: 48, M4K4_sg2: 35, DMSO: 22, GNE 0.1 μM: 34, GNE 0.5 μM: 26, GNE 1.0 μM: 33), from three independent experiments. (D, E) z-scan projection of representative confocal images of F-actin stained A431 clusters, showing the differences in the actin cytoskeleton organization and in the morphology of clusters control (sgNT) or KO for MAP4K4 (M4K4_sg2) or (E) clusters treated with DMSO or GNE-495 at 1.0 μM for 24 h. Arrows represent the actin arches at protrusion bases and arrowheads indicate retraction fibers. (F) Protrusion area of control/ MAP4K4 KO cells or DMSO/GNE-495–treated cells with indicated doses. At least five clusters per experiment, three protrusions per cluster from three independent experiments were analyzed. (G) Circularity of control/ MAP4K4 KO cell clusters, or clusters treated with DMSO or GNE-495 at indicated doses. At least 25 clusters from three independent experiments were analyzed. (H, I) Mean velocity extension (H) or retraction (I) events at the periphery of the clusters before or after treatment with DMSO or GNE-495 at 1.0 μM, over 5 h of treatment. Number of clusters analyzed (DMSO: 28, GNE 0.1 μM: 26, GNE 0.5 μM: 26, GNE 1.0 μM: 28) from three independent experiments. All the data are presented as mean ± s.d. and tested by Kruskal–Wallis (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: MAP4K4 KO cells were generated using the pLenti.Cas9-blast (#52962; Addgene) construct and the following sgRNA constructs: MAP4K4_sg1 (#76263; Addgene) and MAP4K4_sg2 (#76264; Addgene).

Techniques: Western Blot, Control, Migration, Staining

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: (A) Representative confocal images of pERM staining, at the substrate z focal plane, from clusters treated with DMSO or GNE-495 at 1.0 μM. (B) Quantification of mean intensity of pERM from clusters treated with DMSO or GNE-495 at 1.0 μM. At least 25 clusters from three independent experiments were analyzed. (C, D) Number and (D) length of retraction fibers of clusters treated with DMSO or GNE-495 at 1.0 μM. At least 25 clusters from three independent experiments were analyzed. (E) Representative confocal images of pERM staining at the cell–cell junction z focal plane, from clusters treated with DMSO or GNE-495 at 1.0 μM. (F) Quantification of mean intensity of pERM of clusters treated with DMSO or GNE-495 at 1.0 μM. At least 25 clusters from three independent experiments were analyzed. (G) Representative immunoblotting of moesin and actin from lysates of A431 cells control (Rosa_sg) or KO for MSN using two independent sgRNA sequences ( MSN _sg1, MSN _sg2). (H) Representative confocal images of control or MSN KO clusters stained for zyxin. (I) Number of zyxin-positive focal adhesions in control or MSN KO clusters. (J) Representative confocal z-scan projection of p120 stained cells, control or KO for MSN , showing cell–cell junction morphology. (K) Tortuosity index of cell–cell junction on control or MSN KO cells. At least three junctions of five different clusters per experiment, from three independent experiments were analyzed. All the data are presented as mean ± s.d. and tested by Mann–Whitney test (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: MAP4K4 KO cells were generated using the pLenti.Cas9-blast (#52962; Addgene) construct and the following sgRNA constructs: MAP4K4_sg1 (#76263; Addgene) and MAP4K4_sg2 (#76264; Addgene).

Techniques: Staining, Western Blot, Control, MANN-WHITNEY

Journal: Cell death & disease

Article Title: HGK-sestrin 2 signaling-mediated autophagy contributes to antitumor efficacy of Tanshinone IIA in human osteosarcoma cells.

doi: 10.1038/s41419-018-1016-9

Figure Lengend Snippet: Fig. 6 HGK acted an upstream regulator that mediated initiation of the JNK MAP kinase and SESN2-dependent autophagy following TIIA treatment. a Western-assisted analysis of HGK after TIIA treatment as indicated for 24 h in 143B and MG63 cells. b 143B cells were pretreated with GNE-495 (8 nM, 1 h) followed by TIIA treatment as indicated for 24 h. Total lysates were immunoblotted for LC3B, HGK, and p-SAPK/JNK expression. c, d shRNA HGK was stably transfected into 143B (c) and MG63 cells (d). Following treatment with TIIA (20 μM) for indicated time intervals, total lysates were immunoblotted for LC3B, HGK, SESN2, p-SAPK/JNK, JNK1, p-c-Jun, and total c-Jun expression. β-actin served as loading control. e, f Representative images of colonies of 143B-HGKKD (shHGK) and 143B-mock (nonsense) cells in a soft agar colony formation assay in the absence or presence of various concentrations of TIIA were captured using a microscope. Scale bar: 500 μm (e). Results were expressed as average number of colonies counted (in six microfields) (f). g, h 143B cells were transiently transfected with the AP-1 luciferase reporter construct (g) or SESN2 promoter luciferase reporter construct (h). After 24 h, the cells were treated with various concentrations of TIIA for another 12 h and the relative luciferase activity was measured and presented as relative AP-1 activity or relative SESN2 promoter activity. The results were expressed as the means ± SD from three independent experiments (n ≥3, *P < 0.05 compared with untreated control)

Article Snippet: For knockdown of SESN2, BECN1, and HGK, pGFP-VRS plasmid encoding shRNA against human SESN2 (TG301755), BECN1 (TG314484), and HGK (TG320615) were purchased from OriGene (Rockville, MD).

Techniques: Western Blot, Expressing, shRNA, Stable Transfection, Transfection, Control, Soft Agar Assay, Microscopy, Luciferase, Construct, Activity Assay