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: Oncogene

Article Title: MAP4K4 promotes ovarian cancer metastasis through diminishing ADAM10-dependent N-cadherin cleavage

doi: 10.1038/s41388-023-02650-5

Figure Lengend Snippet: A Diagram for tissue collection and data analysis. B Volcano plot shows differentially expressed kinases in peritoneal metastatic tumors as compared to primary tumor based on the diagram in Fig. 1 A . C Western blot analysis of MAP4K4 protein levels in 12 paired ovarian cancer primary and metastatic tissues. D The relative mRNA level of MAP4K4 of 24 pairs of ovarian cancer primary and metastatic tissues. E Representative images of immunohistochemistry staining for MAP4K4 in ovarian cancer tissues. F H-score of IHC staining against MAP4K4 in paired specimen. G Kaplan–Meier plot of overall survival for ovarian cancer patients from local cohort stratified by the MAP4K4 expression level. H The correlation between MAP4K4 expression and CA125 serum level. I The correlation between MAP4K4 expression and ascites volume. J Association between clinicopathological characteristics and MAP4K4 expression.

Article Snippet: Membranes were incubated overnight with primary antibodies, including MAP4K4 (#3485, 1:1000, CST), ADAM10 (#14194, 1:1000, CST), N-cadherin (#13116, 1:1000, CST; 22018-1-AP, 1:1000, Proteintech), vimentin (#5741, 1:1000, CST), pan Phospho-Serine/Threonine Rabbit Polyclonal Antibody (AF5725, 1:1000, Beyotime), HA (#3724, 1:1000, CST), FLAG (#14793, 1:1000, CST), β-actin (AF2811, 1:5000, Beyotime).

Techniques: Western Blot, Immunohistochemistry, Staining, Expressing

Journal: Oncogene

Article Title: MAP4K4 promotes ovarian cancer metastasis through diminishing ADAM10-dependent N-cadherin cleavage

doi: 10.1038/s41388-023-02650-5

Figure Lengend Snippet: A Wound healing assays of control SKOV3 and MAP4K4 knock-down SKOV3 cells were performed every 24 h after the cells were scratched. The histograms on the right show the quantitative results of healing percentage. B Wound healing assays of vehicle control and 5 uM MAP4K4 inhibitor GEN-495 treated SKOV3 cells were performed. Photos were taken every 24 h after the cells were scratched. The histograms on the right show the quantitative results of healing percentage. C Transwell assay for evaluating the migration and invasion of MAP4K4 cells with MAP4K4 knock down in SKOV3 cells. The histograms on the right show the quantitative results of the number of cells per fields. D Representative images of the cell adhesion assay comparing control SKOV3 and MAP4K4 knock down SKOV3 cells. The histograms on the right show the relative 590 nm OD values. n = 5. E Representative images of ZsGreen-labeled SKOV3 cells adhering to primary mesothelial cells. The histograms on the right show the relative fluorescence intensity. F Transwell assay for evaluating the migration of MAP4K4-KO SKOV3 cells and parent cells. The histograms on the right show the quantitative results of the number of cells per fields. G Transwell assay for evaluating the migration of MAP4K4-KO SKOV3 cells with MAP4K4 WT or mutant overexpression. The histograms on the right show the quantitative results of the number of cells per fields. H Representative images of the cell adhesion assay in MAP4K4-KO SKOV3 cells and parent cells. The histograms on the right show the relative 590 nm OD values. I Representative images of the cell adhesion assay in MAP4K4-KO SKOV3 cells with MAP4K4 WT or mutant over-expression. The histograms on the right show the relative 590 nm OD values. J Wound healing assays of A2780 cells with MAP4K4 WT or mutant over-expression. Photos were taken every 24 h after the cells were scratched. The histograms on the right show the quantitative results of healing percentage. Data are presented as the mean ± SD values. Statistical significance was assessed by unpaired t -test was performed. * P < 0.05, ** P < 0.01, *** P < 0.001, n = 3 otherwisely stated.

Article Snippet: Membranes were incubated overnight with primary antibodies, including MAP4K4 (#3485, 1:1000, CST), ADAM10 (#14194, 1:1000, CST), N-cadherin (#13116, 1:1000, CST; 22018-1-AP, 1:1000, Proteintech), vimentin (#5741, 1:1000, CST), pan Phospho-Serine/Threonine Rabbit Polyclonal Antibody (AF5725, 1:1000, Beyotime), HA (#3724, 1:1000, CST), FLAG (#14793, 1:1000, CST), β-actin (AF2811, 1:5000, Beyotime).

Techniques: Control, Knockdown, Transwell Assay, Migration, Cell Adhesion Assay, Labeling, Fluorescence, Mutagenesis, Over Expression

Journal: Oncogene

Article Title: MAP4K4 promotes ovarian cancer metastasis through diminishing ADAM10-dependent N-cadherin cleavage

doi: 10.1038/s41388-023-02650-5

Figure Lengend Snippet: A GSEA results of Metastatic versus Primary tissues. B GSEA enrichment plot of hallmark epithelial mesenchymal transition. C Morphological change in MAP4K4-KO cell with or without TGFβ(2 ng/mL) treatment for 24 h. D MAP4K4 over-expression mimics TGFβ-induced actin organization. Cells were stained for F-actin. TGFβ were applied as 2 ng/mL for 24 h. E Western blot analysis of mesenchymal markers in SKOV3 cells when MAP4K4 were knocked down. F Immunofluorescence assay of the expression level of N-cadherin in SKOV3 cells with MAP4K4 knockdown by siRNA. G Western blot analysis of N-cadherin in SKOV3 cell treated with GNE-495 (2.5, 5, 10 uM for 48 h), or PF-06260933 (5, 10, 20 uM for 48 h). H Western blot analysis of N-cadherin in MAP4K4- KO SKOV3 cells when MAP4K4 wild-type, kinase active mutant or kinase inactive mutant were over-expressed. I The mRNA level of CDH2 (encoding N-cadherin) in the indicated conditions. J Western blot analysis of N-cadherin expression. SKOV3 cells were subjected to MAP4K4 siRNA combined with MG132 as indicated concentrations for 6 h. K Western blot analysis of N-cadherin expression. SKOV3 cells were subjected to MAP4K4 siRNA combined with CQ as indicated concentrations for 6 h. * P < 0.05, ** P < 0.01, *** P < 0.001, n = 3 otherwisely stated.

Article Snippet: Membranes were incubated overnight with primary antibodies, including MAP4K4 (#3485, 1:1000, CST), ADAM10 (#14194, 1:1000, CST), N-cadherin (#13116, 1:1000, CST; 22018-1-AP, 1:1000, Proteintech), vimentin (#5741, 1:1000, CST), pan Phospho-Serine/Threonine Rabbit Polyclonal Antibody (AF5725, 1:1000, Beyotime), HA (#3724, 1:1000, CST), FLAG (#14793, 1:1000, CST), β-actin (AF2811, 1:5000, Beyotime).

Techniques: Over Expression, Staining, Western Blot, Immunofluorescence, Expressing, Knockdown, Mutagenesis

Journal: Oncogene

Article Title: MAP4K4 promotes ovarian cancer metastasis through diminishing ADAM10-dependent N-cadherin cleavage

doi: 10.1038/s41388-023-02650-5

Figure Lengend Snippet: A Relative mRNA expression of genes encoding metalloproteinases (MMPs and ADAMs) in SKOV3 cells. mRNA expression was normalized to the level of β-actin. B , C Representative WB results of N-cadherin when MAP4K4 and other MMPs or ADAMs were knocked down. D Heatmap and cluster dendrogram of relative level of N-cadherin. The signal intensity in WB results of N-cadherin in triple duplication assays were quantified and normalized. E Western blot analysis of the effect of ADAM10 inhibitor GI254023X to N-cadheirn in MAP4K4 knock-down SKOV3 cells. GI254023X was used with the indicated concentration for 12 h. F Molecular modelling to ADAM10/N-cadherin interaction. G Molecular modelling to ADAM10/MAP4K4 interaction. H The free energy change in indicated complexes. I Control cell and siMAP4K4 cells were lysated and immunoprecipitated by anti-ADAM10 antibody. Indicated proteins were checked. J Control cells and MAP4K4 -KO cells were lysated and immunoprecipitated by anti-ADAM10 antibody. Indicated proteins were checked.

Article Snippet: Membranes were incubated overnight with primary antibodies, including MAP4K4 (#3485, 1:1000, CST), ADAM10 (#14194, 1:1000, CST), N-cadherin (#13116, 1:1000, CST; 22018-1-AP, 1:1000, Proteintech), vimentin (#5741, 1:1000, CST), pan Phospho-Serine/Threonine Rabbit Polyclonal Antibody (AF5725, 1:1000, Beyotime), HA (#3724, 1:1000, CST), FLAG (#14793, 1:1000, CST), β-actin (AF2811, 1:5000, Beyotime).

Techniques: Expressing, Western Blot, Knockdown, Concentration Assay, Control, Immunoprecipitation

Journal: Oncogene

Article Title: MAP4K4 promotes ovarian cancer metastasis through diminishing ADAM10-dependent N-cadherin cleavage

doi: 10.1038/s41388-023-02650-5

Figure Lengend Snippet: A Immunofluorescence analysis of the localization of MAP4K4 and ADAM10 in SKOV3 cells. B Endogenous immunoprecipitation in SKOV3 cell with anti-MAP4K4 antibody. C Endogenous immunoprecipitation in SKOV3 cell with anti-ADAM10 antibody. D Flag-MAP4K4 and ADAM10-HA were expressed in HEK293T cells, and immunoprecipitated by anti-Flag antibody. E Flag-MAP4K4 and ADAM10-HA were expressed in HEK293T cells, and immunoprecipitated by anti-HA antibody. F , G MAP4K4 binds to the ADAM10 by CNH domain. The diagram on the right illustrate the interaction between MAP4K4 deletion mutants and ADAM10. H , I ADAM10 binds to the MAP4K4 by cysteine-rich domain. The diagram on the right illustrate the interaction between ADAM10 deletion mutants and MAP4K4. J Western blot with phos-tag gel to dissolve phosphorylated ADAM10 after MAP4K4 knock down. K SKOV3 cells were transfected with indicated expression vectors. The cell extracts were subjected to phos-tag gel to dissolve phosphorylated ADAM10. L SKOV3 cells were transfected with indicated expression vectors. The cell extracts were subjected to immunoprecipitate HA-tagged ADAM10 and immunoblotted with anti-pan Phospho-Serine/Threonine antibody. The whole cell lysates were blotted with indicated antibodies. M Western blot with phos-tag gel to dissolve phosphorylated ADAM10 after treatment with GNE-495 (5 uM for 48 h), or PF-06260933 (10 uM for 48 h). N SKOV3 cells were treated with GNE-495 (5 uM for 48 h) or PF-06260933 (10 uM for 48 h). The cell extracts were subjected to immunoprecipitate HA-tagged ADAM10 and immunoblotted with anti-pan Phospho-Serine/Threonine antibody. The whole cell lysates were blotted with indicated antibodies.

Article Snippet: Membranes were incubated overnight with primary antibodies, including MAP4K4 (#3485, 1:1000, CST), ADAM10 (#14194, 1:1000, CST), N-cadherin (#13116, 1:1000, CST; 22018-1-AP, 1:1000, Proteintech), vimentin (#5741, 1:1000, CST), pan Phospho-Serine/Threonine Rabbit Polyclonal Antibody (AF5725, 1:1000, Beyotime), HA (#3724, 1:1000, CST), FLAG (#14793, 1:1000, CST), β-actin (AF2811, 1:5000, Beyotime).

Techniques: Immunofluorescence, Immunoprecipitation, Western Blot, Knockdown, Transfection, Expressing

Journal: Oncogene

Article Title: MAP4K4 promotes ovarian cancer metastasis through diminishing ADAM10-dependent N-cadherin cleavage

doi: 10.1038/s41388-023-02650-5

Figure Lengend Snippet: A HEK293T cells were transfected with indicated expression vectors. The whole cell lysates were loaded to phos-tag gels to resolve the phosphor-ADAM10 proteins, and then immunoblotted with anti-HA antibody. B HEK293T cells were transfected with indicated expression vectors. The cell extracts were subjected to immunoprecipitate HA-tagged ADAM10 and immunoblotted with anti-pan Phospho-Serine/Threonine antibody. The whole cell lysates were blotted with indicated antibodies. C The potential ADAM10 phosphorylation sites and the corresponding score. D Sequence conservation analysis of the relevant amino acids of ADAM10. E , F HEK-293 cells were transfected with indicated expression vectors. The whole cell lysates were loaded to phos-tag gels to resolve the phosphor-ADAM10 proteins, and then immunoblotted with anti-HA antibody. G , H HEK293T cells were transfected with indicated expression vectors. The whole cell lysates were loaded to phos-tag gels to resolve the phosphor-ADAM10 proteins, and then immunoblotted with anti-HA antibody. I The effect of ADAM10 mutant on N-cadherin expression level. J in vitro kinase assay showing S436A mutant could attenuated ADAM10 phosphorylation by MAP4K4 compared with ADAM10 WT. K Phosphorylated S436 residues in ADAM10 detected by mass spectrometry analysis of SKOV3 cells.

Article Snippet: Membranes were incubated overnight with primary antibodies, including MAP4K4 (#3485, 1:1000, CST), ADAM10 (#14194, 1:1000, CST), N-cadherin (#13116, 1:1000, CST; 22018-1-AP, 1:1000, Proteintech), vimentin (#5741, 1:1000, CST), pan Phospho-Serine/Threonine Rabbit Polyclonal Antibody (AF5725, 1:1000, Beyotime), HA (#3724, 1:1000, CST), FLAG (#14793, 1:1000, CST), β-actin (AF2811, 1:5000, Beyotime).

Techniques: Transfection, Expressing, Phospho-proteomics, Sequencing, Mutagenesis, In Vitro, Kinase Assay, Mass Spectrometry

Journal: Oncogene

Article Title: MAP4K4 promotes ovarian cancer metastasis through diminishing ADAM10-dependent N-cadherin cleavage

doi: 10.1038/s41388-023-02650-5

Figure Lengend Snippet: A Schematic representation of the protocol. B , C Representative pictures of metastasis in nude mouse xenograft models. D HE staining of tumors on the surfaces of liver and spleen. E The metastatic tumors were separated, and photos were taken. F Quantitative analysis of the number of metastatic tumors in mice treated with Vehicle or GNE-495. G Representative images of IHC analyses of tumor specimens from xenograft model using anti-N-cadherin antibody. H Representative images of immunofluorescence of tumor specimens from xenograft model using anti-N-cadherin antibody. I Schematic representation of the protocol. J Representative pictures of metastasis in nude mouse xenograft models. K Representative pictures of tumors. L Quantitative analysis of the number of metastatic tumors in mice. M Schematic representation of ADAM10 S436 phosphorylation by MAP4K4. Phosphorylated ADAM10 contribute N-cadherin stability and therefore promotes ovarian cancer metastasis. Data are presented as the mean ± SD values. Statistical significance was assessed by unpaired t -test. * P < 0.05, ** P < 0.01, *** P < 0.001, n = 5.

Article Snippet: Membranes were incubated overnight with primary antibodies, including MAP4K4 (#3485, 1:1000, CST), ADAM10 (#14194, 1:1000, CST), N-cadherin (#13116, 1:1000, CST; 22018-1-AP, 1:1000, Proteintech), vimentin (#5741, 1:1000, CST), pan Phospho-Serine/Threonine Rabbit Polyclonal Antibody (AF5725, 1:1000, Beyotime), HA (#3724, 1:1000, CST), FLAG (#14793, 1:1000, CST), β-actin (AF2811, 1:5000, Beyotime).

Techniques: Staining, Immunofluorescence, Phospho-proteomics

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: Scientific Reports

Article Title: Overcoming radioresistance of breast cancer cells with MAP4K4 inhibitors

doi: 10.1038/s41598-024-57000-6

Figure Lengend Snippet: Overexpression of MAP4K4 in radioresistant breast cancer cell lines derived from SK-BR-3 and MCF-7 cells. (A) Clonogenic survival assays after irradiating cells with 1, 2, 3, 4, or 5 Gy. Radioresistant SK-BR-3 (SR) cells and radioresistant MCF-7 (MR) cells were resistant to radiation as compared with parental cells (SK-BR-3 and MCF-7). (B) Western blot revealed MAP4K4 expression was markedly elevated in SR and MR cells. MAP4K4 expression levels are expressed as fold changes versus parental cells. (C) Immunofluorescence analysis of MAP4K4 in parental (SK-BR-3 and MCF-7) and radioresistant (SR and MR) cells . (D) Western blot analysis of p-ERK 1/2, ERK 1/2, p-JNK 1/2, JNK 1/2, p-p38, and p38 in SR and MR cells versus parental cells. p-ERK 1/2, ERK 1/2, p-JNK 1/2, JNK 1/2, p-p38, and p38 protein levels are expressed as fold changes versus parental cells. Graphs were plotted using the means ± SDs of three independent experiments. * and ** indicate P values of < 0.05 and P < 0.01 versus parental cells, respectively.

Article Snippet: Primary antibodies for acyl-CoA synthetase-4 (ACSL4), RAD51, and β-actin, and siRNA for MAP4K4 (sc-39243), ACSL4 (sc-60619), and control (sc-37007) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Over Expression, Derivative Assay, Western Blot, Expressing, Immunofluorescence

Journal: Scientific Reports

Article Title: Overcoming radioresistance of breast cancer cells with MAP4K4 inhibitors

doi: 10.1038/s41598-024-57000-6

Figure Lengend Snippet: MAP4K4 inhibitors (GNE-495 and PF06260933) selectively targeted radioresistant cells. (A) Effects of PF06260933 and GNE-495 on MAP4K4 kinase activities. (B) Western blot of MAP4K4 in SR cells treated with PF06260933 or GNE-495. MAP4K4 protein levels are expressed as fold changes versus untreated cells (0 µM). Cytotoxic effects of (C) PF06260933 and (D) GNE-495 on parental (SK-BR-3 and MCF-7) and radioresistant (SR and MR) cells. Cells were treated with PF06260933 or GNE-495 for 48 h and cell viabilities were assessed using the sulforhodamine B (SRB) assay. Results are presented as means ± SDs, ** indicates P values of < 0.01 versus untreated cells.

Article Snippet: Primary antibodies for acyl-CoA synthetase-4 (ACSL4), RAD51, and β-actin, and siRNA for MAP4K4 (sc-39243), ACSL4 (sc-60619), and control (sc-37007) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Western Blot, Sulforhodamine B Assay

Journal: Scientific Reports

Article Title: Overcoming radioresistance of breast cancer cells with MAP4K4 inhibitors

doi: 10.1038/s41598-024-57000-6

Figure Lengend Snippet: MAP4K4 inhibitor suppressed tumor growth in mice bearing SR tumor. (A) The experimental scheme used for testing the anti-tumor effects of PF06260933 (PF) in vivo. (B) No loss of body weight occurred in mice treated with PF06260933. (C) Tumor growth in SR tumor-bearing BALB/c nude mice treated with PF06260933 (n = 4). (D) Final tumor volume on day of sacrifice. * indicates P value of < 0.05 versus control group.

Article Snippet: Primary antibodies for acyl-CoA synthetase-4 (ACSL4), RAD51, and β-actin, and siRNA for MAP4K4 (sc-39243), ACSL4 (sc-60619), and control (sc-37007) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: In Vivo, Control

Journal: Scientific Reports

Article Title: Overcoming radioresistance of breast cancer cells with MAP4K4 inhibitors

doi: 10.1038/s41598-024-57000-6

Figure Lengend Snippet: Targeting MAP4K4 suppressed DNA damage response and induced apoptosis in radioresistant cells. (A) Expressions of p-p38, p-H2AX, and RAD51 in SR cells treated with PF06260933 or GNE-495 for 24 h. p-p38, p-H2AX, and RAD51 levels are expressed as fold changes versus untreated cells (0 µM). (B) Expressions of apoptotic markers, cleaved caspase 3, and cleaved PARP, and survivin in SR cells treated with PF06260933 or GNE-495 for 24 h. Cleaved caspase 3, cleaved PARP, and survivin levels are expressed as fold changes versus untreated cells (0 µM). Results are presented as means ± SDs, and * and ** indicate p -values of < 0.05 and < 0.01 versus untreated cells, respectively.

Article Snippet: Primary antibodies for acyl-CoA synthetase-4 (ACSL4), RAD51, and β-actin, and siRNA for MAP4K4 (sc-39243), ACSL4 (sc-60619), and control (sc-37007) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques:

Journal: Scientific Reports

Article Title: Overcoming radioresistance of breast cancer cells with MAP4K4 inhibitors

doi: 10.1038/s41598-024-57000-6

Figure Lengend Snippet: MAP4K4 functioned as an upstream effector of ACSL4. (A) Immunofluorescence analysis of MAP4K4 and ACSL4 in SR and MR cells indicated that MAP4K4 co-localized with ACSL4 in radioresistant cells. (B) Expressions of ACSL4 in SR cells treated with PF06260933 or GNE-495. (C) Expressions of ACSL4 and MAP4K4 in SR cells treated with triacsin C (ACSL4 inhibitor). (D) Effect of triacsin C on MAP4K4 kinase activity. The protein expressions of MAP4K4 and ACSL4 following siRNA knockdown. MAP4K4 (E) and ACSL4 (F) protein levels are expressed as fold changes versus untreated cells (0 µM). Results are presented as means ± SDs, and * and ** indicate P values of < 0.05 and < 0.01, respectively.

Article Snippet: Primary antibodies for acyl-CoA synthetase-4 (ACSL4), RAD51, and β-actin, and siRNA for MAP4K4 (sc-39243), ACSL4 (sc-60619), and control (sc-37007) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Immunofluorescence, Activity Assay, Knockdown

Journal: Scientific Reports

Article Title: Overcoming radioresistance of breast cancer cells with MAP4K4 inhibitors

doi: 10.1038/s41598-024-57000-6

Figure Lengend Snippet: Targeting MAP4K4 overcame the radioresistances of SR and MR cells and suppressed their metastatic properties. (A) Radioresistance of SR and MR cells after exposure to 2 Gy/day for 5 days. SR and MR cells were highly resistant to 2 Gy/day × 5 radiation as compared with parental cells. Clonogenic survival assays of SR and MR cells exposed to 2 Gy/day for 5 days in presence or absence of (B) PF06260933 (PF) or (C) GNE-495 (GNE). Results are presented as means ± SDs, and * and ** indicate P values of < 0.05 and < 0.01, respectively. (D) Transwell invasion assays of SR and MR cells treated with 1 μM PF06260933 or 10 nM GNE-495 for 24 h.

Article Snippet: Primary antibodies for acyl-CoA synthetase-4 (ACSL4), RAD51, and β-actin, and siRNA for MAP4K4 (sc-39243), ACSL4 (sc-60619), and control (sc-37007) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques:

Journal: Scientific Reports

Article Title: Overcoming radioresistance of breast cancer cells with MAP4K4 inhibitors

doi: 10.1038/s41598-024-57000-6

Figure Lengend Snippet: Schematic diagram of the suggested MAP4K4-ACSL4 signaling axis in radioresistant breast cancer cells. We suggest MAP4K4 positively regulates ACSL4 to mediate radioresistance by enhancing DNA damage response and inhibiting apoptosis.

Article Snippet: Primary antibodies for acyl-CoA synthetase-4 (ACSL4), RAD51, and β-actin, and siRNA for MAP4K4 (sc-39243), ACSL4 (sc-60619), and control (sc-37007) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: