anti map4k4  (Proteintech)

Journal: PLOS Computational Biology

Article Title: Machine learning-based prediction reveals kinase MAP4K4 regulates neutrophil differentiation through phosphorylating apoptosis-related proteins

doi: 10.1371/journal.pcbi.1012877

Figure Lengend Snippet: (A) Three-component Gaussian Mixture Models (GMM) were applied to the NeuRGI scores across all 19,288 predicted genes. The solid black line represents the overall score distribution, dashed lines indicate the three probability density functions (PDFs). Intersections of the three PDFs define NeuRGI thresholds for classifying genes into function (red shadow), non-function (blue shadow), and uncertain (middle white) categories. (B) The line plot depicts the overlap of functional transcription factors (TFs) predicted by CellOracle and NeuRGI (red line), and by SCENIC and NeuRGI (blue line). The x-axis represents the top N TFs ranked by CellOracle’s perturbation score or SCENIC regulon activity. The y-axis shows the percentage of overlap between the predicted functional TFs. (C) The boxplot illustrates the transformed feature values for 4,786 functional genes and 4,734 non-functional genes in four feature groups. The p value was calculated using the Student’s t-test. (D) The GO-BP term network illustrates the five main clusters enriched from 4,786 predictive functional genes (see Methods). Each dot represents a GO term and the dot size indicates the enrichment score. The dashed oval indicates GO terms with similar functions. (E) Proportion of regulatory (red) and other (blue) genes across three gene sets. Regulatory genes include enzyme (Enz), membrane protein (MP), RNA binding protein (RBP), and transcription factor (TF). The p value was calculated by the proportion test. Numbers within bars represent the gene counts. (F) Boxplot of NeuRGI scores, color-coded by gene type: Enzyme (Enz), membrane proteins (MP), RNA binding proteins (RBP), Transcription factors (TF), and others. The p value was calculated using the Student’s t-test. (G) The scatter plot shows the impact of in silico knockout of 2,569 predictive functional regulatory genes on the “positive regulation of myeloid cell differentiation” pathway and MAGMA Zscore (neutrophil count). The y-axis represents the -log 10 (p value) on the pathway after gene in silico knockout (higher values indicate greater impact), and the x-axis represents the gene’s effect on the ‘neutrophil count’ trait (higher Zscores indicate greater impact). Different colors represent different categories of genes. Dot size indicates the NeuRGI score, and contour lines show point density. A cutoff (y = 1.8 and x = 3.6) was set based on the contour lines, dividing the scatter plot into four regions. (H) Expression of top 12 genes in different immune cells from ImmuNexUT, including Naïve CD4 T cells (Naïve CD4), Memory CD4 T cells (Mem CD4), T helper 1 cells (Th1), T helper 2 cells (Th2), T helper 17 cells (Th17), T follicular helper cells (Tfh), Fraction II effector regulatory T cells (Fr. II eTreg), Fraction I naïve regulatory T cells (Fr. I nTreg), Fraction III non-regulatory T cells (Fr. III T), Naïve CD8 T cells (Naïve CD8), CD8+ T effector memory CD45RA+ cells (TEMRA CD8), Effector Memory CD8 T cells (EM CD8), Central Memory CD8 T cells (CM CD8), Naïve B cells (Naïve B), Unswitched memory B cells (USM B), Switched memory B cells (SM B), Double Negative B cells (DN B), Plasmablasts (Plasmablast), Natural Killer cells (NK), CD16 positive monocytes (CD16p Mono), Non-classical monocytes (NC Mono), Intermediate monocytes (Int Mono), Classical monocytes (CL Mono), Myeloid dendritic cells (mDC), Plasmacytoid dendritic cells (pDC), neutrophils (Neu), Low-Density Granulocytes (LDG). (I) Expression of 9 genes in neutrophil differentiation of human UCB. These 9 of the top 12 genes dynamically upregulated, including CREBBP , CYP27A1 , JAK2 , IFNGR1 , MAP4K4 , PLCG2 , PTPRC, TIGAR, and TYK2 . We set ‘time cut’ for cells at different differentiation stages, with HSC set as 1 and Neu as 5, and performed linear regression fitting for the expression of all 9 genes. R represents the Pearson correlation coefficient (R), and the p value (p) was calculated by the Student’s t-test. (J) Expression of top 10 genes in single-cell pseudotime analysis of 2,803 neutrophils in mouse bone marrow. All 12 genes except Cyp27a1 and HLA-DQA1 upregulated during neutrophil maturation. (K) The heatmap displays the log 2 (fold change) in the expression of the top 12 genes in neutrophils from patients with 10 immune-related diseases compared to those from healthy individuals, including ANCA-associated vasculitis (AAV), Takayasu arteritis (TAK), Adult-onset Still’s disease (AOSD), Behçet’s disease (BD), Rheumatoid arthritis (RA), Systemic sclerosis (SSc), Idiopathic inflammatory myopathy (Myo), Sjögren’s syndrome (SjS), Mixed connective tissue disease (MCTD), Systemic lupus erythematosus (SLE). The p value was calculated using the Student’s t-test, and * represents statistical significance (p < 0.05). The histogram represents the number of significant diseases for each gene. (L) Bar plot displaying significantly affected pathways after OntoVAE in silico knockout of MAP4K4 in neutrophils. The blue dashed line represents the significance threshold (p = 0.05).

Article Snippet: The blots were probed with the primary antibodies including rabbit anti-MAP4K4 antibody (CST, #3485), rabbit anti-p-STAT5A (Tyr699) antibody (Abcam, # ab32043), rabbit monoclonal (E289) anti-STAT5A antibody (Abcam, #ab32043), rabbit anti-STAT5B antibody (Abcam, #ab30648), rabbit monoclonal (C11C5) anti-p-STAT5 (Tyr694) antibody (CST, #9359), rabbit monoclonal (D2O6Y) anti-STAT5 antibody (CST, #94205), rabbit monoclonal (14C10) anti-GAPDH antibody (Cell Signaling Technology, #2118) in the universal antibody diluent (NCM biotech, #WB500D) for overnight at 4°C, washed three times with TBST, and then incubated with the HRP conjugated goat anti-rabbit antibodies (HuaBio, #HA1001).

Techniques: Functional Assay, Activity Assay, Transformation Assay, Membrane, RNA Binding Assay, In Silico, Knock-Out, Cell Differentiation, Expressing

Journal: PLOS Computational Biology

Article Title: Machine learning-based prediction reveals kinase MAP4K4 regulates neutrophil differentiation through phosphorylating apoptosis-related proteins

doi: 10.1371/journal.pcbi.1012877

Figure Lengend Snippet: (A) Scheme of all-trans-retinoic acid (ATRA)-induced neutrophil differentiation of the hematopoietic HL-60 cell models (left); flow cytometry analysis of the neutrophil marker CD11b has validated that ATAR can induce differentiation of HL-60 cells into neutrophils. This is demonstrated by histogram plots showing variations in mean fluorescence intensity (MFI) across different conditions: cells untreated with ATAR, cells treated with ATAR for 72 hours, and cells treated with ATAR for 96 hours (right). (B) Percentage of CD11b + cells in HL-60 cells were treated with 0-20 µM MAP4K4 inhibitor (PF-06260933, PF) for 48 hours, followed by a subsequent 96-hour treatment with both PF and all-trans-retinoic acid (ATRA). (C) The number of granulocyte-macrophage progenitor cells (CFU-GM, CFU-G, CFU-M) colonies formed by 25,000 whole bone marrow cells from control (Ctrl) or Map4k4 -cKO (cKO) mice; BM, bone marrow (n=4; mean ± SD). (D) Numbers of neutrophils in the bone marrow of control (Ctrl) or Map4k4 -cKO (cKO) mice; BM, bone marrow; Neu, neutrophil. (Ctrl n=7, cKO n=6; mean ± SD). (E) PB WBC numbers of control (Ctrl) or Map4k4 -cKO (cKO) mice (Ctrl n=7, cKO n=6; mean ± SD). PB, peripheral blood; WBC, white blood cell; (Ctrl n=7, cKO n=6; mean ± SD). (F) PB Mon, and GR numbers of control (Ctrl) or Map4k4 -cKO (cKO) mice; PB, peripheral blood; Mon, monocyte; GR, granulocyte; (Ctrl n=7, cKO n=6; mean ± SD). (G) Numbers of eosinophils in PB of control (Ctrl) or Map4k4 -cKO (cKO) mice; PB, peripheral blood; Eso, eosinophils; (Ctrl n=7, cKO n=6; mean ± SD). (H) Numbers of monocytes in PB of control (Ctrl) or Map4k4 -cKO (cKO) mice; PB, peripheral blood; Mon, monocytes; (Ctrl n=7, cKO n=6; mean ± SD). (I) Numbers of neutrophils in PB of control (Ctrl) or Map4k4 -cKO (cKO) mice; PB, peripheral blood; Neu, neutrophil; (Ctrl n=7, cKO n=6; mean ± SD). (J) Spleen cell numbers control (Ctrl) or Map4k4 -cKO (cKO) mice (Ctrl n=7, cKO n=6; mean ± SD). (K) Numbers of eosinophils in the spleen of control (Ctrl) or Map4k4 -cKO (cKO) mice; SP, spleen; Eso, eosinophils; (Ctrl n=7, cKO n=6; mean ± SD). (L) Numbers of monocytes in the spleen of control (Ctrl) or Map4k4 -cKO (cKO) mice; SP, spleen; Mon, monocytes; (Ctrl n=7, cKO n=6; mean ± SD). (M) Representative FACS analysis of spleen CD11b+ Ly6G+ neutrophils (left); percentage of neutrophils in the spleen of control (Ctrl) or Map4k4 -cKO (cKO) mice (right); SP, spleen; Neu, neutrophil; (Ctrl n=7, cKO n=6; mean ± SD). (N) Numbers of neutrophils in the spleen of control (Ctrl) or Map4k4 -cKO (cKO) mice; SP, spleen; Neu, neutrophil; (Ctrl n=7, cKO n=6; mean ± SD). Mann-Whitney U test.

Article Snippet: The blots were probed with the primary antibodies including rabbit anti-MAP4K4 antibody (CST, #3485), rabbit anti-p-STAT5A (Tyr699) antibody (Abcam, # ab32043), rabbit monoclonal (E289) anti-STAT5A antibody (Abcam, #ab32043), rabbit anti-STAT5B antibody (Abcam, #ab30648), rabbit monoclonal (C11C5) anti-p-STAT5 (Tyr694) antibody (CST, #9359), rabbit monoclonal (D2O6Y) anti-STAT5 antibody (CST, #94205), rabbit monoclonal (14C10) anti-GAPDH antibody (Cell Signaling Technology, #2118) in the universal antibody diluent (NCM biotech, #WB500D) for overnight at 4°C, washed three times with TBST, and then incubated with the HRP conjugated goat anti-rabbit antibodies (HuaBio, #HA1001).

Techniques: Flow Cytometry, Marker, Fluorescence, Control, MANN-WHITNEY

Journal: PLOS Computational Biology

Article Title: Machine learning-based prediction reveals kinase MAP4K4 regulates neutrophil differentiation through phosphorylating apoptosis-related proteins

doi: 10.1371/journal.pcbi.1012877

Figure Lengend Snippet: (A) Overview of study design, created using Biorender. (B) UMAP Plots for single-cell gene expression pooled across bone marrow samples, clusters visualized and labeled by cell type. The plot is split by control (Ctrl) vs Map4k4 -cKO (cKO). (C) Proportions of the six neutrophil clusters in control (Ctrl) and Map4k4 -cKO (cKO) samples. The p value was calculated by the adjusted proportion test. (D) Numbers of neutrophil progenitor cells in the bone marrow of control (Ctrl) or Map4k4 -cKO (cKO) mice; c-Kit hi Ly6G neg (myeloblasts, MB); c-Kit int Ly6G neg (promyelocytes, PM); c-Kit neg Ly6G low (myelocytes, MC); c-Kit neg Ly6G int (metamyelocytes, MM); and c-Kit neg Ly6G hi (band cells and segmented neutrophils, BC/SC); BM, bone marrow (Ctrl n=7, cKO n=6; mean ± SD). Mann-Whitney U test. (E) ROS generation of neutrophil progenitor cells in BM of control (Ctrl) or Map4k4 -cKO (cKO) mice; MFI, mean fluorescent intensity; ROS, reactive oxygen species; MB, myeloblasts; PM, promyelocytes; MC, myelocytes; MM, metamyelocytes; BC/SC, band cells and segmented neutrophils; (n=6; mean ± SD). Mann-Whitney U test. (F) The number of DEGs in control (Ctrl) vs Map4k4 -cKO (cKO) of six neutrophil subpopulations. (G) GO-BP analysis of cluster-based DEGs between control (Ctrl) and Map4k4 -cKO (cKO) HSC to neutrophils. Selected GO terms with Benjamini-Hochberg-corrected p values < 0.05 (one-sided Fisher’s exact test) are shown. The dot size represented the number of genes. The color scale represented the adjusted p value. was created using Biorender.

Article Snippet: The blots were probed with the primary antibodies including rabbit anti-MAP4K4 antibody (CST, #3485), rabbit anti-p-STAT5A (Tyr699) antibody (Abcam, # ab32043), rabbit monoclonal (E289) anti-STAT5A antibody (Abcam, #ab32043), rabbit anti-STAT5B antibody (Abcam, #ab30648), rabbit monoclonal (C11C5) anti-p-STAT5 (Tyr694) antibody (CST, #9359), rabbit monoclonal (D2O6Y) anti-STAT5 antibody (CST, #94205), rabbit monoclonal (14C10) anti-GAPDH antibody (Cell Signaling Technology, #2118) in the universal antibody diluent (NCM biotech, #WB500D) for overnight at 4°C, washed three times with TBST, and then incubated with the HRP conjugated goat anti-rabbit antibodies (HuaBio, #HA1001).

Techniques: Gene Expression, Labeling, Control, MANN-WHITNEY

Journal: PLOS Computational Biology

Article Title: Machine learning-based prediction reveals kinase MAP4K4 regulates neutrophil differentiation through phosphorylating apoptosis-related proteins

doi: 10.1371/journal.pcbi.1012877

Figure Lengend Snippet: (A) UMAP plot of the neutrophil co-expression network. Each node represents a single gene, and edges represent co-expression links between genes and module hub genes. Point size is scaled by eigengene-based connectivity (kME). Nodes are colored by co-expression module assignment. The top five hub genes per module are labeled. Network edges were down-sampled for visual clarity. (B) Heatmap of scaled gene expression for the top 10 hub genes by kME in each module. (C) UMAP colored by Pseudotime. (D) UMAP colored by harmonized module eigengenes (hMEs) of each module. (E) Module eigengenes (MEs) as a function of pseudotime for each co-expression module. For each module, a separate loess regression line is shown for each condition. The p value was calculated by distance correlation t-test (dcorT.test). dcor is a transformation of a bias-corrected version of distance correlation. The smaller the dcor, the greater the distance between control (Ctrl) and Map4k4 -cKO (cKO) conditions. (F) Differentially expressed hub genes between control (Ctrl) and Map4k4 -cKO (cKO) parasites involved in positive regulation of apoptotic process determined by Gene Set Enrichment Analysis (GSEA). RES represents the running enrichment score. RLM represents the ranked list metric. (G) Violin plots of apoptosis scores of control (Ctrl) against Map4k4 -cKO (cKO) for each cluster, the p value was calculated by the Student’s t-test. (H) Percentages of annexin V + and SYTOX − cells in bone marrow neutrophil progenitor cells of control (Ctrl) or Map4k4 -cKO (cKO) mice; MM, metamyelocytes; BC/SC, band cells and segmented neutrophils; BM, bone marrow (n=10; mean ± SD). Mann-Whitney U test. (I) Percentages of annexin V + and SYTOX − cells of HL-60 cells treated with 0-20 µM MAP4K4 inhibitor (PF-06260933, PF) for 48 hours, followed by a subsequent 96-hour treatment with both PF and all-trans-retinoic acid (ATRA) (n=4; mean ± SD). Mann-Whitney U test. (J) Quantification of HL-60 cell numbers after a 48-hour treatment with a 10 µM concentration of the MAP4K4 inhibitor (PF-06260933, PF), followed by a subsequent 96-hour treatment with both PF and all-trans-retinoic acid (ATRA) (n=4; mean ± SD). Mann-Whitney U test.

Article Snippet: The blots were probed with the primary antibodies including rabbit anti-MAP4K4 antibody (CST, #3485), rabbit anti-p-STAT5A (Tyr699) antibody (Abcam, # ab32043), rabbit monoclonal (E289) anti-STAT5A antibody (Abcam, #ab32043), rabbit anti-STAT5B antibody (Abcam, #ab30648), rabbit monoclonal (C11C5) anti-p-STAT5 (Tyr694) antibody (CST, #9359), rabbit monoclonal (D2O6Y) anti-STAT5 antibody (CST, #94205), rabbit monoclonal (14C10) anti-GAPDH antibody (Cell Signaling Technology, #2118) in the universal antibody diluent (NCM biotech, #WB500D) for overnight at 4°C, washed three times with TBST, and then incubated with the HRP conjugated goat anti-rabbit antibodies (HuaBio, #HA1001).

Techniques: Expressing, Labeling, Gene Expression, Transformation Assay, Control, MANN-WHITNEY, Concentration Assay

Journal: PLOS Computational Biology

Article Title: Machine learning-based prediction reveals kinase MAP4K4 regulates neutrophil differentiation through phosphorylating apoptosis-related proteins

doi: 10.1371/journal.pcbi.1012877

Figure Lengend Snippet: (A) Schematic of mass spectrometry analysis, created using Biorender. (B) The number of proteins with significant differences in protein and phosphorylation levels after MAP4K4 knockout. (C) Comparison heatmap of log 2 (fold-change) in protein and phosphorylation levels after MAP4K4 knockout. (D) Differential phosphorylation level between control and MAP4K4 KO parasites is involved in the positive regulation of the apoptotic process, as determined by Gene Set Enrichment Analysis (GSEA). RES represents the running enrichment score. RLM represents the ranked list metric. (E) Volcano plot of the apoptosis-related proteins with differentially expressed phosphorylation levels. The x-axis shows the phosphorylation difference of log 2 (fold-change) between MAP4K4 KO and Ctrl. The y-axis shows –log 10 (adjusted p value) of the t-test between these two groups. Red indicates proteins with up-regulated phosphorylation after MAP4K4 KO, and blue, proteins with down-regulated phosphorylation after MAP4K4 KO. The top 20 proteins with the most significant differences in phosphorylation among the apoptosis-related proteins are visualized. (F) Western blot (WB) for MAP4K4, p-STAT5A, STAT5A, p-STAT5B, STAT5B, and GAPDH in sg AASV1 and sg MAP4K4 cells. (G) Representative FACS analysis of HL-60 cells were first infected with overexpression of STAT5A or STAT5B or control plasmids and then treated with 10 µM MAP4K4 inhibitor (PF-06260933, PF) for 48 hours, followed by a subsequent 96-hour treatment with both PF and all-trans-retinoic acid (ATRA) for staining of anti–annexin V and SYTOX was shown (left). Percentages of annexin V + and SYTOX − cells (right) (n=4; mean ± SD). Mann-Whitney U test. was created with Biorender.

Article Snippet: The blots were probed with the primary antibodies including rabbit anti-MAP4K4 antibody (CST, #3485), rabbit anti-p-STAT5A (Tyr699) antibody (Abcam, # ab32043), rabbit monoclonal (E289) anti-STAT5A antibody (Abcam, #ab32043), rabbit anti-STAT5B antibody (Abcam, #ab30648), rabbit monoclonal (C11C5) anti-p-STAT5 (Tyr694) antibody (CST, #9359), rabbit monoclonal (D2O6Y) anti-STAT5 antibody (CST, #94205), rabbit monoclonal (14C10) anti-GAPDH antibody (Cell Signaling Technology, #2118) in the universal antibody diluent (NCM biotech, #WB500D) for overnight at 4°C, washed three times with TBST, and then incubated with the HRP conjugated goat anti-rabbit antibodies (HuaBio, #HA1001).

Techniques: Mass Spectrometry, Phospho-proteomics, Knock-Out, Comparison, Control, Western Blot, Infection, Over Expression, Staining, MANN-WHITNEY

Journal: bioRxiv

Article Title: The Cardioprotective Effect of Ischemic Postconditioning is Mediated by Inhibiting RAP2C-MAP4K4 Pathway

doi: 10.1101/2024.12.04.626922

Figure Lengend Snippet: a. Protein interactions based on the analysis of the BioGRID and STRING databases. b. Immunohistochemical analysis of MAP4K4 expression in rat myocardial tissue exposed to sham, I/R and I/R-postC in rat myocardial tissue (scale bar = 25 µm). c. MAP4K4 expression in rat cardiomyocytes exposed to sham conditions, ischemia/reperfusion (I/R), or I/R + PostC. d. MAP4K4 expression in rat cardiomyocytes exposed to normoxia (N), hypoxia/reoxygenation (H/R), or H/R + PostC. e. MAP4K4 expression in cardiomyocytes transfected with si-RAP2C or si-NC and exposed to H/R or H/R + PostC. f. MAP4K4 expression in cardiomyocytes transduced with Ad-RAP2C or Ad-null and exposed to H/R or H/R + PostC. g. Co-immunoprecipitation assays using cardiomyocyte lysates and RAP2C antibody under normoxia, H/R, and H/R + PostC. MAP4K4 and RAP2C expression in input and immunoprecipitated samples was evaluated by western blotting. h. Colocalization of MAP4K4 and RAP2C by immunofluorescence double staining (scale bar = 5/10 μm). The degree of colocalization is shown in scatter plots and was calculated using Pearson’s correlation coefficient. Data are means and standard deviations of three biological replicates. p values were calculated by two-tailed Student’s t- test. one-way ANOVA with Tukey’s post hoc test was used for multiple group comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Myocardial tissue was fixed in 4% paraformaldehyde for 10 min and permeabilized in Triton X-100 1% for 20 min. Endogenous peroxidase activity was blocked using 5% BSA for 1 h. Tissues were incubated with primary antibodies against RAP2C (orb474405, Biorbyt, Cambridge, UK) and MAP4K4 (sc-100445, Santa Cruz) overnight at 4℃, followed by incubation with CoraLite594-conjugated goat anti-mouse antibody (SA00013-3, Proteintech) and CoraLite488-conjugated goat anti-rabbit antibody (SA00013-2, Proteintech) at room temperature for 1 h. Nuclei were stained with DAPI.

Techniques: Immunohistochemical staining, Expressing, Transfection, Transduction, Immunoprecipitation, Western Blot, Immunofluorescence, Double Staining, Two Tailed Test

Journal: bioRxiv

Article Title: The Cardioprotective Effect of Ischemic Postconditioning is Mediated by Inhibiting RAP2C-MAP4K4 Pathway

doi: 10.1101/2024.12.04.626922

Figure Lengend Snippet: a. Western blot analysis of the efficiency of siRNA-mediated silencing of MAP4K4 expression. b. p-ERK, p-JNK, and p-P38 expression in cardiomyocytes transfected with si-MAP4K4 or si-NC and exposed to normoxia (N), H/R, or H/R + PostC. c. Number of TUNEL-positive cells in different groups (×10) (scale bar = 200 µm). d. Flow cytometry analysis of total apoptosis rates in different groups. e. Western blot analysis of the expression of cleaved caspase-3 and −9 and the Bax/Bcl-2 ratio. Data are means and standard deviations of three biological replicates. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Myocardial tissue was fixed in 4% paraformaldehyde for 10 min and permeabilized in Triton X-100 1% for 20 min. Endogenous peroxidase activity was blocked using 5% BSA for 1 h. Tissues were incubated with primary antibodies against RAP2C (orb474405, Biorbyt, Cambridge, UK) and MAP4K4 (sc-100445, Santa Cruz) overnight at 4℃, followed by incubation with CoraLite594-conjugated goat anti-mouse antibody (SA00013-3, Proteintech) and CoraLite488-conjugated goat anti-rabbit antibody (SA00013-2, Proteintech) at room temperature for 1 h. Nuclei were stained with DAPI.

Techniques: Western Blot, Expressing, Transfection, TUNEL Assay, Flow Cytometry

Journal: bioRxiv

Article Title: The Cardioprotective Effect of Ischemic Postconditioning is Mediated by Inhibiting RAP2C-MAP4K4 Pathway

doi: 10.1101/2024.12.04.626922

Figure Lengend Snippet: a. Western blot analysis of RAP2C and MAP4K4 expression in cells transfected with Ad-RAP2C or Ad-RAP2C + si-MAP4K4 and exposed to hypoxia/reoxygenation (H/R) and ischemic postconditioning (PostC). b. Protein expression of p-ERK, p-JNK, and p-P38 in different groups. c. Number of TUNEL-positive cells in different groups (×10) (scale bar = 100 µm). d. Flow cytometry analysis of the rate of apoptosis. e. Western blot analysis of the Bax/Bcl-2 ratio and the expression of cleaved caspase-3 and −9 in cardiomyocytes. Data are means and standard deviations of three biological replicates. p values were calculated by two-tailed Student’s t- test. one-way ANOVA with Tukey’s post hoc test was used for multiple group comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Myocardial tissue was fixed in 4% paraformaldehyde for 10 min and permeabilized in Triton X-100 1% for 20 min. Endogenous peroxidase activity was blocked using 5% BSA for 1 h. Tissues were incubated with primary antibodies against RAP2C (orb474405, Biorbyt, Cambridge, UK) and MAP4K4 (sc-100445, Santa Cruz) overnight at 4℃, followed by incubation with CoraLite594-conjugated goat anti-mouse antibody (SA00013-3, Proteintech) and CoraLite488-conjugated goat anti-rabbit antibody (SA00013-2, Proteintech) at room temperature for 1 h. Nuclei were stained with DAPI.

Techniques: Western Blot, Expressing, Transfection, TUNEL Assay, Flow Cytometry, Two Tailed Test

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