Journal: bioRxiv

Article Title: Phosphatidylserine-Based Liposomes Encapsulating DMX-5804 Protect Against Doxorubicin-Induced Cardiotoxicity

doi: 10.64898/2026.02.12.705423

Figure Lengend Snippet: Comparative analysis of MAP4K4 inhibitors reveals differential effects on H9c2 cell viability in the presence of doxorubicin. (A) Effect of MAP4K4 Inhibitors on the viability of H9c2 Cells. (B) Schematic illustrating viability studies conducted on H9c2 cells treated with doxorubicin and MAP4K4 inhibitors. (C) Viability of H9c2 Cells Treated with Doxorubicin and DMX-5804. (D) Viability of H9c2 cells treated with doxorubicin and GNE-495, (E) Viability of H9c2 cells treated with doxorubicin and MAP4K4-IN3, (F) Viability of H9c2 cells treated with doxorubicin and PF-06260933. Statistical analysis was performed using two-way ANOVA to assess the effects of treatment and concentration, followed by Sidak’s multiple-comparisons post hoc test to compare groups at matched concentrations. Data are presented as mean ± stdev, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: The following primary antibodies were used: MAP4K4 (rabbit IgG, Proteintech catalog no. 55247-1-AP, 1:1000 dilution), phospho-MAP4K4 (Ser629) (rabbit IgG, Bioss, catalog no. BS-5491R, 1:2000 dilution), cJUN (rabbit IgG, catalog no. 1:1000 dilution), phospho-cJUN (Ser73) (rabbit IgG, Cell Signaling Technology catalog no. 3270, 1:1000), and beta-actin, HRP (rabbit IgG, Invitrogen, catalog no. PA1-183-HRP, 1:1000) dilution.

Techniques: Concentration Assay

Journal: bioRxiv

Article Title: Phosphatidylserine-Based Liposomes Encapsulating DMX-5804 Protect Against Doxorubicin-Induced Cardiotoxicity

doi: 10.64898/2026.02.12.705423

Figure Lengend Snippet: Effects of DMX-5804 on doxorubicin-induced gene expression, drug uptake, and protein signaling in H9c2 cardiomyoblasts. (A) qPCR analysis of H9c2 cells treated with doxorubicin, DMX-5804, or both for 4 hours. (B) qPCR analysis of H9c2 cells treated with doxorubicin, DMX-5804, or both for 24 hours. RT-qPCR data are shown as mean ± SD from n = 3 independent biological replicates, each measured in duplicate technical replicates. (C) Uptake of 10 μM doxorubicin into H9c2 cells co-treated with 10 μM MAP4K4 inhibitors (DMX-5804, GNE-495, MAP4K4-IN3, and PF-06260933) after 24 hours. Data represent mean ± standard deviation from six technical replicates per condition from a single experiment; statistical comparisons were performed using one-way ANOVA. (D) Uptake of 10 μM doxorubicin into H9c2 cells co treated with increasing concentrations of DMX-5804 (5 μM, 10 μM, and 20 μM) after 24 hours. Data represent mean ± standard deviation from six technical replicates per condition from a single experiment; statistical comparisons were performed using one-way ANOVA followed by Dunnett’s multiple comparison’s post-test. (E) Western blot analysis of H9c2 cells treated with doxorubicin, DMX-5804, or both for 24 hours and 4 hours. Membranes were probed for β-actin as a loading control to verify equal protein loading across lanes. Blots shown are representative of at least three independent experiments.

Article Snippet: The following primary antibodies were used: MAP4K4 (rabbit IgG, Proteintech catalog no. 55247-1-AP, 1:1000 dilution), phospho-MAP4K4 (Ser629) (rabbit IgG, Bioss, catalog no. BS-5491R, 1:2000 dilution), cJUN (rabbit IgG, catalog no. 1:1000 dilution), phospho-cJUN (Ser73) (rabbit IgG, Cell Signaling Technology catalog no. 3270, 1:1000), and beta-actin, HRP (rabbit IgG, Invitrogen, catalog no. PA1-183-HRP, 1:1000) dilution.

Techniques: Gene Expression, Quantitative RT-PCR, Standard Deviation, Western Blot, Control

Journal: bioRxiv

Article Title: Phosphatidylserine-Based Liposomes Encapsulating DMX-5804 Protect Against Doxorubicin-Induced Cardiotoxicity

doi: 10.64898/2026.02.12.705423

Figure Lengend Snippet: Effects of MAP4K4 inhibition on doxorubicin uptake, cell viability, and protein signaling in breast cancer cells. (A) Effect of MAP4K4 inhibitors on the cell viability of MDA-MB-231 cells. (B) Uptake of doxorubicin into MDA-MB-231 cells co-treated with MAP4K4 inhibitors after 24 hours. (C) Viability of MDA-MB-231 cells treated with doxorubicin and DMX-5804. (D) Viability of 4T1 cells treated with Doxorubicin and DMX-5804. (E) Western blot analysis of MDA-MB-231 cells treated with doxorubicin, DMX-5804, or both for 24 hours. Data are presented as mean ± stdev, n=4. Statistical analysis was performed using two-way ANOVA to assess the effects of treatment and concentration, followed by Sidak’s multiple-comparisons post hoc test to compare groups at matched concentrations. Data are presented as mean ± stdev, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: The following primary antibodies were used: MAP4K4 (rabbit IgG, Proteintech catalog no. 55247-1-AP, 1:1000 dilution), phospho-MAP4K4 (Ser629) (rabbit IgG, Bioss, catalog no. BS-5491R, 1:2000 dilution), cJUN (rabbit IgG, catalog no. 1:1000 dilution), phospho-cJUN (Ser73) (rabbit IgG, Cell Signaling Technology catalog no. 3270, 1:1000), and beta-actin, HRP (rabbit IgG, Invitrogen, catalog no. PA1-183-HRP, 1:1000) dilution.

Techniques: Inhibition, Western Blot, Concentration Assay

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: Advanced Science

Article Title: Peptide Transporter 1‐Mediated Dipeptide Transport Promotes Hepatocellular Carcinoma Metastasis by Activating MAP4K4/G3BP2 Signaling Axis

doi: 10.1002/advs.202306671

Figure Lengend Snippet: PEPT1‐mediated HCC metastasis was dependent on MAP4K4. A) Protein expression of EMT‐associated proteins in HCC cells with PEPT1 overexpression or knockdown. B) Volcano plot of all differential genes in Huh7 cells that stably express shRNA stargeting PEPT1 or scramble control. C) Go analysis showed that differentially expressed genes were mainly enriched in protein kinase binding. D) The protein expression of MAP4K4 in PEPT1‐overexpression or PEPT1‐silencing HCC cells was detected by Western blot analysis. E) MAP4K4 protein levels in fresh HCC and adjacent nontumor tissues detection by Western blot ( n = 12). F) Representative IHC images of MAP4K4 in HCC tissue ( n = 10) and corresponding normal tissue ( n = 10). Scale bar, 100 µm. G) The correlation between PEPT1 and MAP4K4 was analyzed based on HCC date from the ICJC (LIRI‐JP) database (left) and Western blot results (right). H) Kaplan–Meier analysis of overall survival (OS) data from ICJC (LIRI‐JP) liver cancer database. I) Representative images and quantification of the indicated cells in the wound‐healing migration assays. Scale bar, 100 µm. J) Representative images and quantification of the migration and invasion of the indicated cells in the Transwell assays. Scale bar, 250 µm. K) Protein expression of EMT‐associated proteins in HCC cells with MAP4K4 knockdown. L) Representative images and quantification of the migration and invasion of the indicated cells in the Transwell assays. Scale bar, 250 µm. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: [ ] Briefly, after the indicated treatments, the cells were cultured in a 15 mm glass‐bottom confocal dish for 24 h. The cells were then washed with phosphate buffered saline (PBS), fixed with 4% paraformaldehyde for 15 min, and permeabilized with 0.5% Triton X‐100 for 20 min. After blocking, the slides were staining overnight at 4 °C with indicated primary antibodies: HA (1:50, Proteintech, #51064‐2‐AP), Flag (1:2000, Proteintech, #66008‐4‐Ig), and MAP4K4 (1:50, Cusabio, #CSB‐PA013439LA01HU).

Techniques: Expressing, Over Expression, Knockdown, Stable Transfection, shRNA, Control, Binding Assay, Western Blot, Migration

Journal: Advanced Science

Article Title: Peptide Transporter 1‐Mediated Dipeptide Transport Promotes Hepatocellular Carcinoma Metastasis by Activating MAP4K4/G3BP2 Signaling Axis

doi: 10.1002/advs.202306671

Figure Lengend Snippet: MAP4K4 directly binds to G3BP2 in HCC. A) Phosphorylated proteomics analysis identified G3BP2 in the binding protein pool. B,C) Endogenous interaction between MAP4K4 and G3BP2 was determined using co‐IP with anti‐MAP4K4 or anti‐G3BP2 antibodies in Huh7 and PLC/PRF/5 cells. D) Exogenous interaction between MAP4K4 and G3BP2 was determined using co‐IP with anti‐Flag or anti‐HA antibodies in HEK 293T cells co‐transfected with Flag‐G3BP2 and HA‐MAP4K4. E) Immunofluorescence staining showing colocalization of endogenous MAP4K4 (red) and G3BP2 (green) in Huh7 and PLC/PRF/5 cells. The nucleus is labeled by DAPI (blue). Scale bar: 20 µm. F) Immunofluorescence staining showing colocalization of exogenous HA‐MAP4K4 (red) and Flag‐G3BP2 (green) in HEK293T cells. The nucleus is labeled by DAPI (blue). Scale bar: 20 µm. G) Representative IHC images for MAP4K4 and G3BP2 in pulmonary metastatic lesions of nude mice developed by PEPT1‐knockdown or PEPT1‐overexpression HCC cells. Scale bar, 500 µm, 50 µm.

Article Snippet: [ ] Briefly, after the indicated treatments, the cells were cultured in a 15 mm glass‐bottom confocal dish for 24 h. The cells were then washed with phosphate buffered saline (PBS), fixed with 4% paraformaldehyde for 15 min, and permeabilized with 0.5% Triton X‐100 for 20 min. After blocking, the slides were staining overnight at 4 °C with indicated primary antibodies: HA (1:50, Proteintech, #51064‐2‐AP), Flag (1:2000, Proteintech, #66008‐4‐Ig), and MAP4K4 (1:50, Cusabio, #CSB‐PA013439LA01HU).

Techniques: Binding Assay, Co-Immunoprecipitation Assay, Transfection, Immunofluorescence, Staining, Labeling, Knockdown, Over Expression

Journal: Advanced Science

Article Title: Peptide Transporter 1‐Mediated Dipeptide Transport Promotes Hepatocellular Carcinoma Metastasis by Activating MAP4K4/G3BP2 Signaling Axis

doi: 10.1002/advs.202306671

Figure Lengend Snippet: MAP4K4‐mediated phosphorylation at T227 is required for the function of G3BP2 in HCC metastasis. A) HEK293T cells were transfected with vectors, Flag‐G3BP2 along with HA‐MAP4K4. The cell extracts were used to immunoprecipitated Flag‐G3BP2 and blotted with anti‐p‐Ser/Thr/Tyr and anti‐Flag antibodies. The whole‐cell lysate (WCL) was blotted with anti‐HA, anti‐Flag, and anti‐β‐actin antibodies. B) The MAP4K4 knockdown Huh7 and PLC/PRF/5 extracts were used to immunoprecipitated G3BP2 and blotted with anti‐p‐Ser/Thr/Tyr and anti‐G3BP2 antibodies. The WCL was blotted with anti‐MAP4K4, anti‐G3BP2, and anti‐β‐actin antibodies. C) Schematic diagram of G3BP2 structure and phosphorylation sites. D) HEK293T cells were transfected with Flag‐G3BP2 (WT), Flag‐G3BP2 (T227A), or HA‐MAP4K4 as the indicated combinations. The cell extracts were used to immunoprecipitated Flag‐G3BP2 and blotted with anti‐p‐Ser/Thr/Tyr and anti‐Flag antibodies. The WCL was blotted with anti‐HA, anti‐Flag and anti‐β‐actin antibodies. E) Huh7 and PLC/PRF/5 cells were transfected with vector, G3BP2 (WT) or G3BP2 (T227A), and the protein expression of MAP4K4, G3BP2, EMT‐associated proteins were detected by Western blot. F) Representative images and quantification of the migration and invasion of the indicated cells in the Transwell assays. Scale bar, 250 µm. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: [ ] Briefly, after the indicated treatments, the cells were cultured in a 15 mm glass‐bottom confocal dish for 24 h. The cells were then washed with phosphate buffered saline (PBS), fixed with 4% paraformaldehyde for 15 min, and permeabilized with 0.5% Triton X‐100 for 20 min. After blocking, the slides were staining overnight at 4 °C with indicated primary antibodies: HA (1:50, Proteintech, #51064‐2‐AP), Flag (1:2000, Proteintech, #66008‐4‐Ig), and MAP4K4 (1:50, Cusabio, #CSB‐PA013439LA01HU).

Techniques: Phospho-proteomics, Transfection, Immunoprecipitation, Knockdown, Plasmid Preparation, Expressing, Western Blot, Migration

Journal: Advanced Science

Article Title: Peptide Transporter 1‐Mediated Dipeptide Transport Promotes Hepatocellular Carcinoma Metastasis by Activating MAP4K4/G3BP2 Signaling Axis

doi: 10.1002/advs.202306671

Figure Lengend Snippet: G3BP2 was upregulated in HCC and influenced cell migration and invasion. A) G3BP2 protein levels in fresh HCC and adjacent nontumor tissues detected by Western blot ( n = 12). B) Representative IHC images of G3BP2 in HCC tissue ( n = 10) and corresponding normal tissue ( n = 10). Scale bar, 100 µm. C) The correlation between PEPT1 and G3BP2 (left), and MAP4K4 and G3BP2 (right) was analyzed based on the Western blot results. D) Representative images and quantification of the indicated cells in the wound‐healing migration assays. Scale bar, 100 µm. E) Representative images and quantification of the migration and invasion of the indicated cells in the Transwell assays. Scale bar, 250 µm. F) Protein expression of EMT‐associated proteins in HCC cells with G3BP2 knockdown. G) Representative images and quantification of the migration and invasion of the indicated cells in the Transwell assays. Scale bar, 250 µm. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: [ ] Briefly, after the indicated treatments, the cells were cultured in a 15 mm glass‐bottom confocal dish for 24 h. The cells were then washed with phosphate buffered saline (PBS), fixed with 4% paraformaldehyde for 15 min, and permeabilized with 0.5% Triton X‐100 for 20 min. After blocking, the slides were staining overnight at 4 °C with indicated primary antibodies: HA (1:50, Proteintech, #51064‐2‐AP), Flag (1:2000, Proteintech, #66008‐4‐Ig), and MAP4K4 (1:50, Cusabio, #CSB‐PA013439LA01HU).

Techniques: Migration, Western Blot, Expressing, Knockdown

Journal: Advanced Science

Article Title: Peptide Transporter 1‐Mediated Dipeptide Transport Promotes Hepatocellular Carcinoma Metastasis by Activating MAP4K4/G3BP2 Signaling Axis

doi: 10.1002/advs.202306671

Figure Lengend Snippet: PEPT1‐mediated dipeptides transport was essential for activating MAP4K4/G3BP2 axis. A) Endogenous interaction between PEPT1 and MAP4K4 was determined using co‐IP with anti‐MAP4K4 antibodies in Huh7 and PLC/PRF/5 cells. B) Metabolomics analysis identified dipeptides downregulated in Huh7 cells with stably PEPT1 knockdown. C) The uptake of Pro‐Gly in HCC cells with PEPT1 overexpressing and knockdown. D) Representative images and quantification of the migration and invasion of the indicated cells in the Transwell assays. Scale bar, 250 µm. E) Huh7 and PLC/PRF/5 cells were incubated with the indicated concentrations of Ile‐Ala or Gln‐Tyr for 24 hours, and the protein expression of MAP4K4, G3BP2, EMT‐associated proteins were detected by Western blot. Error bars indicate means ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: [ ] Briefly, after the indicated treatments, the cells were cultured in a 15 mm glass‐bottom confocal dish for 24 h. The cells were then washed with phosphate buffered saline (PBS), fixed with 4% paraformaldehyde for 15 min, and permeabilized with 0.5% Triton X‐100 for 20 min. After blocking, the slides were staining overnight at 4 °C with indicated primary antibodies: HA (1:50, Proteintech, #51064‐2‐AP), Flag (1:2000, Proteintech, #66008‐4‐Ig), and MAP4K4 (1:50, Cusabio, #CSB‐PA013439LA01HU).

Techniques: Co-Immunoprecipitation Assay, Stable Transfection, Knockdown, Migration, Incubation, Expressing, Western Blot

Journal: Advanced Science

Article Title: Peptide Transporter 1‐Mediated Dipeptide Transport Promotes Hepatocellular Carcinoma Metastasis by Activating MAP4K4/G3BP2 Signaling Axis

doi: 10.1002/advs.202306671

Figure Lengend Snippet: PEPT1/MAP4K4/G3BP2 signaling axis promoted HCC metastasis. A,C) Western blot analysis showed that the knockdown efficacy of MAP4K4 or G3BP2 in Bel7405 and HCCLM3 cells with PEPT1 overexpressing. B,D) Transwell assays revealed that migration and invasion ability was abrogated in Bel7405 and HCCLM3 cells with MAP4K4 or G3BP2 knockdown compared with the control group. Scale bar, 250 µm. E,G) Western blot analysis showed that the overexpression efficacy of G3BP2 (or PEPT1) in Huh7 cells with MAP4K4 (or G3BP2) knockdown. F,H) Inhibited migration and invasion of Huh7 cells with MAP4K4 (or G3BP2) knocked down was rescued by the overexpression of G3BP2 (or PEPT1). Left, representative images of Transwell assays, scale bar, 250 µm. Right, statistical analysis of Transwell assays. I) A schematic diagram of the PEPT1/MAP4K4/G3BP2 regulatory signaling axis that facilitates HCC metastasis.

Article Snippet: [ ] Briefly, after the indicated treatments, the cells were cultured in a 15 mm glass‐bottom confocal dish for 24 h. The cells were then washed with phosphate buffered saline (PBS), fixed with 4% paraformaldehyde for 15 min, and permeabilized with 0.5% Triton X‐100 for 20 min. After blocking, the slides were staining overnight at 4 °C with indicated primary antibodies: HA (1:50, Proteintech, #51064‐2‐AP), Flag (1:2000, Proteintech, #66008‐4‐Ig), and MAP4K4 (1:50, Cusabio, #CSB‐PA013439LA01HU).

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

Journal: PLoS Pathogens

Article Title: The Inflammatory Kinase MAP4K4 Promotes Reactivation of Kaposi's Sarcoma Herpesvirus and Enhances the Invasiveness of Infected Endothelial Cells

doi: 10.1371/journal.ppat.1003737

Figure Lengend Snippet: MAP4K4 was silenced with siRNA in EA.hy rKSHV.219 or HuAR2T rKSHV.219 cells twenty-four hours prior to the induction of the viral lytic cycle. Forty-two hours after activating the lytic cycle with Na-butyrate and RTA transduction (see Material and Methods) cells and supernatants were harvested for subsequent analysis. (A) KSHV titre before and after MAP4K4 knockdown. HEK293 cells were infected with viral supernatants from siRNA treated EA.hy rKSHV.219 cells. Production of infectious virus particles was quantified by counting GFP-positive HEK293 cells forty-eight hours after infection. The bar graph shows means ±SD of five independent experiments. The p value was determined using a One-way ANOVA with Tukey's multiple comparison post-test. p<0.01 (**). (B) Western blot analysis of KSHV lytic protein expression. EA.hy rKSHV.219 cells were lysed, cell extracts resolved by SDS-PAGE and the indicated KSHV proteins have been detected with specific antibodies. The blot is one representative of seven independent experiments with similar results. (C) KSHV genome copy number after MAP4K4 depletion or foscarnet treatment. HuAR2T rKSHV.219 cells were lysed, DNA extracted and KSHV genome copy number was evaluated by qPCR analysis. The bar graph shows means ±SD of three independent experiments. The p value was determined using a One-way ANOVA with Tukey's multiple comparison post-test. p<0.001 (***). (D) Western blot analysis of KSHV lytic protein expression after MAP4K4 depletion or Foscarnet treatment. HuAR2T rKSHV.219 cells were lysed, cell extracts resolved by SDS-PAGE and the indicated KSHV proteins detected with specific antibodies. The blot is one representative of three independent experiments with similar results.

Article Snippet: MAP4K4 was stained immunohistochemically with MAP4K4 monoclonal antibody M07, clone 4A5, produced by Abnova Corp. and purchased from Biozol Diagnostica GmbH, applied at a 1∶300 dilution.

Techniques: Transduction, Knockdown, Infection, Virus, Comparison, Western Blot, Expressing, SDS Page

Journal: PLoS Pathogens

Article Title: The Inflammatory Kinase MAP4K4 Promotes Reactivation of Kaposi's Sarcoma Herpesvirus and Enhances the Invasiveness of Infected Endothelial Cells

doi: 10.1371/journal.ppat.1003737

Figure Lengend Snippet: Uninfected HuAR2T cells, or HuAR2T cells stably infected with rKSHV.219 were treated with Na-butyrate and a baculovirus vector expressing RTA, or left untreated, for twenty-four hours with subsequent starving for twelve hours in EBM2 basal medium supplemented with 2% FBS. Equal numbers of cells were seeded on growth factor reduced Matrigel invasion chambers. After twenty-four hours invaded cells were fixed and stained with DAPI. (A) Representative images of invasive cells before and after induction of the lytic cycle. (B) Invasion score measured by quantification of DAPI signal from invasive cells. The bar graph shows means ±SD of invasion scores of three independent experiments. (C) Representative images of invasive KSHV-infected cells before and after MAP4K4 depletion and after induction of the lytic replication cycle. MAP4K4 or control siRNA were transfected twenty-four hours prior to the induction of the lytic replication cycle. (D) Invasion score presented as means ±SD of three independent experiments. The p values were determined using a One-way ANOVA with Tukey's multiple comparison post-test. p>0.05 (ns); p<0.05 (*); p<0.01 (**); p<0.001 (***); p<0.0001 (****). (E) Western blot analysis of MAP4K4 and KSHV KbZIP early lytic protein expression.

Article Snippet: MAP4K4 was stained immunohistochemically with MAP4K4 monoclonal antibody M07, clone 4A5, produced by Abnova Corp. and purchased from Biozol Diagnostica GmbH, applied at a 1∶300 dilution.

Techniques: Stable Transfection, Infection, Plasmid Preparation, Expressing, Staining, Control, Transfection, Comparison, Western Blot

Journal: PLoS Pathogens

Article Title: The Inflammatory Kinase MAP4K4 Promotes Reactivation of Kaposi's Sarcoma Herpesvirus and Enhances the Invasiveness of Infected Endothelial Cells

doi: 10.1371/journal.ppat.1003737

Figure Lengend Snippet: HuAR2T rKSHV.219 cells were transfected with control siRNA or siRNA targeting MAP4K4 twenty-four hours before the induction of the lytic cycle. Cells were harvested twenty-four hours after inducing the lytic cycle. (A) Alterations in cellular gene expression following MAP4K4 knockdown and KSHV lytic reactivation. 54 cellular genes regulated by MAP4K4 (columns 4–6; grey scale) by a factor of >1.5 were identified by comparing their expression in lytically induced HuAR2T rKSHV.219 cells silenced for MAP4K4 expression with control siRNA treated induced cells. The effect of lytic KSHV reactivation on cellular gene expression is shown on a red-green scale in cells treated with either control siRNA (‘control/control’) or MAP4K4 siRNA (‘MAP4K4/MAP4K4’) (columns 7–12). Uninfected HuAR2T cells were analysed in two additional experiments to evaluate the effects of the induction compounds in the absence of KSHV (columns 13–14). Data were ordered according to the average fold induction strength in control siRNA treated HuAR2T rKSHV.219 cells after lytic induction (columns 7, 9, 11). The fold ratios for PTGS2 presented in columns 7 to 12, which show induced/uninduced ratios from control siRNA treated samples (columns 7, 9, 11) versus siMAP4K4 treated samples (columns 8, 10, 12), appear to be similar. This is due to the fact that COX-2 expression is reduced by a similar factor in uninduced and induced samples following MAP4K4 silencing. (B) qPCR analysis of COX-2, MMP-7 and MMP-13 expression in HuAR2T rKSHV.219 using dually labelled probes presented as means ±SD of three independent experiments. The p values were determined using a One-way ANOVA with Tukey's multiple comparison post-test. p>0.05 (ns); p<0.05 (*); p<0.01 (**); p<0.001 (***). (C) Western blot analysis of COX-2 expression before and after MAP4K4 depletion in latent and lytically induced HuAR2T rKSHV.219 cells. The blot is one representative of five independent experiments with similar results.

Article Snippet: MAP4K4 was stained immunohistochemically with MAP4K4 monoclonal antibody M07, clone 4A5, produced by Abnova Corp. and purchased from Biozol Diagnostica GmbH, applied at a 1∶300 dilution.

Techniques: Transfection, Control, Gene Expression, Knockdown, Expressing, Comparison, Western Blot

Journal: PLoS Pathogens

Article Title: The Inflammatory Kinase MAP4K4 Promotes Reactivation of Kaposi's Sarcoma Herpesvirus and Enhances the Invasiveness of Infected Endothelial Cells

doi: 10.1371/journal.ppat.1003737

Figure Lengend Snippet: HuAR2T or HuAR2T rKSHV.219 cells were transfected with control siRNA or siRNA pools targeting MAP4K4, MMP7 or MMP13 twenty-four hours before the induction of the lytic cycle. Thirty-six hours after lytic reactivation starved cells were analysed for invasiveness. (A) Invasion score determined by quantifying DAPI stained invasive cells during latency and in the course of lytic reactivation and presented as means ±SD of four independent experiments. The p values were determined using a One-way ANOVA with Tukey's multiple comparison post-test. p>0.05 (ns); p<0.05 (*); p<0.01 (**); p<0.001 (***); p<0.0001 (****). (B) Western blot analysis of MAP4K4 and KbZIP expression in HuAR2T and HuAR2T rKSHV.219 cells. The blot is one representative of four independent experiments with similar results. (C) qPCR analysis of MMP-7 and MMP-13 expression in HuAR2T and HuAR2T rKSHV.219 cells. The graph is one representative of four independent experiments with similar results. (D) qPCR analysis of KbZIP and K8.1 mRNA expression in HuAR2T and HuAR2T rKSHV.219 cells. The graph is one representative of four independent experiments with similar results.

Article Snippet: MAP4K4 was stained immunohistochemically with MAP4K4 monoclonal antibody M07, clone 4A5, produced by Abnova Corp. and purchased from Biozol Diagnostica GmbH, applied at a 1∶300 dilution.

Techniques: Transfection, Control, Staining, Comparison, Western Blot, Expressing

Journal: PLoS Pathogens

Article Title: The Inflammatory Kinase MAP4K4 Promotes Reactivation of Kaposi's Sarcoma Herpesvirus and Enhances the Invasiveness of Infected Endothelial Cells

doi: 10.1371/journal.ppat.1003737

Figure Lengend Snippet: HuAR2T rKSHV.219 cells were transfected with control siRNA, MAP4K4- or COX-2 targeting siRNA pools or treated with NS-398 or vehicle control, and subsequently analysed for invasiveness and KSHV lytic protein expression. (A) Invasion score determined after MAP4K4 or COX-2 depletion by quantifying DAPI stained invasive cells. The graph is one representative of two independent experiments with similar results. (B) Western blot analysis of MAP4K4, COX-2, KbZIP, and K8.1 expression. The blot is one representative of two independent experiments with similar results. (C) Invasion score determined after MAP4K4 depletion or COX-2 chemical inhibition by quantifying DAPI stained invasive cells. The graph is one representative of three independent experiments with similar results. (D) Western blot analysis of KSHV lytic protein expression after MAP4K4 silencing or treatment with COX-2 inhibitor NS-398. The blot is one representative of three independent experiments with similar results. (E) Western blot analysis of KSHV lytic protein expression after treatment with COX-2 inhibitor NS-398. The blot is one representative of three independent experiments with similar results. (F) KSHV titre determined by quantifying GFP positive HEK293 cells forty-eight hours after infection with supernatants from untreated or NS-398 treated HuAR2T rKSHV.219 cells. The graph shows means ±SD of three independent experiments. The p values were determined using a One-way ANOVA with Tukey's multiple comparison post-test. p>0.05 (ns); p<0.05 (*); p<0.01 (**); p<0.001 (***); p<0.0001 (****).

Article Snippet: MAP4K4 was stained immunohistochemically with MAP4K4 monoclonal antibody M07, clone 4A5, produced by Abnova Corp. and purchased from Biozol Diagnostica GmbH, applied at a 1∶300 dilution.

Techniques: Transfection, Control, Expressing, Staining, Western Blot, Inhibition, Infection, Comparison

Journal: PLoS Pathogens

Article Title: The Inflammatory Kinase MAP4K4 Promotes Reactivation of Kaposi's Sarcoma Herpesvirus and Enhances the Invasiveness of Infected Endothelial Cells

doi: 10.1371/journal.ppat.1003737

Figure Lengend Snippet: HUVEC were infected with concentrated rKSHV.219 at an MOI of 20 and monitored for MAP4K4 and KSHV lytic protein expression and invasiveness. (A) Representative images of invasive uninfected or rKSHV.219-infected HUVEC before and after MAP4K4 depletion. (B) Invasion score determined by quantifying DAPI stained uninfected or rKSHV.219-infected HUVEC, before and after MAP4K4 depletion. The graph shows means ±SD of three independent experiments. The p values were determined using a One-way ANOVA with Tukey's multiple comparison post-test. p>0.05 (ns); p<0.05 (*); p<0.01 (**); p<0.001 (***); p<0.0001 (****). (C) Western blot analysis of MAP4K4, COX-2 and LANA expression in uninfected or infected HUVEC. The blot is one representative of three independent experiments with similar results. (D) Western blot analysis of MAP4K4, LANA, ORF45 and KbZIP protein expression after infection of HUVEC with rKSHV.219 and MAP4K4 depletion.

Article Snippet: MAP4K4 was stained immunohistochemically with MAP4K4 monoclonal antibody M07, clone 4A5, produced by Abnova Corp. and purchased from Biozol Diagnostica GmbH, applied at a 1∶300 dilution.

Techniques: Infection, Expressing, Staining, Comparison, Western Blot

Journal: PLoS Pathogens

Article Title: The Inflammatory Kinase MAP4K4 Promotes Reactivation of Kaposi's Sarcoma Herpesvirus and Enhances the Invasiveness of Infected Endothelial Cells

doi: 10.1371/journal.ppat.1003737

Figure Lengend Snippet: (A) Subepidermal Kaposi's sarcoma [KS] surrounding and infiltrating a non-neoplastic blood vessel [V]. The tumour cells express CD34 (red; the non-neoplastic endothelial cells of the blood vessel are CD34 positive, too) and nuclear LANA (dark brown), as well as cytoplasmic MAP4K4 (light red; double-staining). (B) Kaposi's sarcoma completely replacing the lymphatic tissue of a lymph node. Nuclear LANA protein expression is observed in 50–70% of tumour cells (dark-brown) and cytoplasmic MAP4K4 expression – in more than 95% of sarcoma cells (brown). (C) Kaposi's sarcoma [KS] partly expressing LANA and infiltrating non-neoplastic subepidermal connective tissue [CT] with a non-neoplastic blood vessel [V]. Strong cytoplasmic MAP4K4 positivity is seen in KS cells, while only weak MAP4K4 expression is present in non-neoplastic endothelial cells [V]. All images are presented in 250× magnification. (D) Summary of MAP4K4 expression in 13 KS biopsies.

Article Snippet: MAP4K4 was stained immunohistochemically with MAP4K4 monoclonal antibody M07, clone 4A5, produced by Abnova Corp. and purchased from Biozol Diagnostica GmbH, applied at a 1∶300 dilution.

Techniques: Double Staining, Expressing