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: Frontiers in Pharmacology

Article Title: Silybum marianum peptide-rich fraction alleviates acute liver injury by inhibiting TRAF3/NIK/NF-κB and is associated with changes in gut microbiota composition

doi: 10.3389/fphar.2026.1915943

Figure Lengend Snippet: SMP protected against CCl 4 -induced liver injury in mice by inhibiting NIK/NF-κB. 8-Week-old male C57BL/6J mice were divided into the following five groups: the control group (treated daily with saline and an intraperitoneal injection of olive oil on the 14th day), CCl 4 group (treated daily with saline and an intraperitoneal injection of CCl 4 dissolved in olive oil on the 14th day), SMP group (treated daily with SMP at 50, 100, or 200 mg/kg, and an intraperitoneal injection of CCl 4 dissolved in olive oil on the 14th day). (A) Immunofluorescence observation of macrophages in liver tissue (F4/80); (B) Immunofluorescence observation of neutrophils in liver tissue (Ly6G); (C) Immunofluorescence observation of CD8 T cells in liver tissue (CD8 + ); (D) Protein expression levels of NRF2 determined via Western blot; (E) Protein expression levels of TRAF3 determined via Western blot; (F) Protein expression levels of NIK, and p52 determined via Western blot. All data are presented as the mean ± SD. Statistical significance was determined by one-way ANOVA followed by the Student–Newman–Keuls post hoc test. For immunofluorescence and Western blot analyses, quantitative data were derived from independent biological samples from individual mice. n = 8, *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Then, primary antibodies against NIK (#55247-1-AP, Proteintech), p52 (#15503-1-AP, Proteintech), NRF2 (#80593-1-RR, Proteintech), GAPDH (#60004-1-Ig, Proteintech), and TRAF3 (#SAB3500376-100UG, Sigma-Aldrich) were added at a dilution of 1:2000 and incubated ove,rnight at 4 °C.

Techniques: Control, Saline, Injection, Immunofluorescence, Tissue, Expressing, Western Blot, Derivative Assay

Journal: Frontiers in Pharmacology

Article Title: Silybum marianum peptide-rich fraction alleviates acute liver injury by inhibiting TRAF3/NIK/NF-κB and is associated with changes in gut microbiota composition

doi: 10.3389/fphar.2026.1915943

Figure Lengend Snippet: SMP alleviated cell damage by inhibiting NIK/NF-κB. After the appropriate SMP concentration (0, 0.01, 0.1, 1, 10, 20, 40, 80, or 160 μg/mL) was determined in normal Hepa1-6 cells, the effect of SMP on the expression of inflammation-induced marker genes was evaluated in Hepa1-6 cells overexpressing the NIK gene. Hepa1-6 cells were divided into the following five groups (n = 3): the oe-vector group (overexpressing vector and administered saline), the solvent group (overexpressing the NIK gene and administered solvent), and the SMP group (overexpressing the NIK gene and administered SMP at concentrations of 10, 20, or 40 μg/mL). (A) CCK8 assay was used to measure the viability of normal Hepa1-6 cells; (B) Protein expression levels of NRF2, NIK, and p52 determined via Western blot; (C) Relative dual luciferase activity indicating transcription activity; (D) mRNA expression levels of TNF-α, CCL2, and CCL5, as detected by qPCR; (E) Cytokine levels, including those of TNF-α, CCL2, and CCL5, were detected via ELISA; (F) The mRNA expression levels of CXCL5 and NOS2 were detected by qPCR. Data are presented as the mean ± SD from three independent experiments (n = 3), with three technical replicates per independent experiment. Statistical significance was determined by one-way ANOVA followed by the Student–Newman–Keuls post hoc test. N = 3, *P < 0.05, **P < 0.01, ***P < 0.001. SMP, Silybum marianum peptides.

Article Snippet: Then, primary antibodies against NIK (#55247-1-AP, Proteintech), p52 (#15503-1-AP, Proteintech), NRF2 (#80593-1-RR, Proteintech), GAPDH (#60004-1-Ig, Proteintech), and TRAF3 (#SAB3500376-100UG, Sigma-Aldrich) were added at a dilution of 1:2000 and incubated ove,rnight at 4 °C.

Techniques: Concentration Assay, Expressing, Marker, Plasmid Preparation, Saline, Solvent, CCK-8 Assay, Western Blot, Luciferase, Activity Assay, Enzyme-linked Immunosorbent 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: 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: BMC Biotechnology

Article Title: In silico screening and in vitro biological evaluation reveal Queuine as a promising MAP4K4 inhibitor for treating pancreatic cancer

doi: 10.1186/s12896-025-01033-w

Figure Lengend Snippet: Interaction analysis of the control drug Gemcitabine and the proposed nutraceutical therapeutics Queuine and Thiamine with the MAP4K4 binding site. ( A ) 3D binding pose of Gemcitabine (left) and 2D representation (right) of Gemcitabine docked in the MAP4K4 binding site. ( B ) 3D and 2D binding pose of Queuine ( C ) 3D and 2D binding pose of Thiamine. Coloring schemes: hydrogen bonding (purple arrows), pi-pi stacking (green line), and pi-cation interactions (red line)

Article Snippet: The ELISA was performed following the protocol provided by the Human Mitogen-activated Protein Kinase 4 (MAP4K4) ELISA Kit, which is intended for the quantitative detection of MAP4K4 (BT Lab, Cat. no. E5689Hu) with slight modifications.

Techniques: Control, Binding Assay

Journal: BMC Biotechnology

Article Title: In silico screening and in vitro biological evaluation reveal Queuine as a promising MAP4K4 inhibitor for treating pancreatic cancer

doi: 10.1186/s12896-025-01033-w

Figure Lengend Snippet: Protein-ligand complex root mean square deviation (RMSD) ( A ) and root mean square fluctuation (RMSF) ( B ) plots for the apo, Gemcitabine, Queuine, and Thiamine bound forms of the MAP4K4 protein

Article Snippet: The ELISA was performed following the protocol provided by the Human Mitogen-activated Protein Kinase 4 (MAP4K4) ELISA Kit, which is intended for the quantitative detection of MAP4K4 (BT Lab, Cat. no. E5689Hu) with slight modifications.

Techniques:

Journal: BMC Biotechnology

Article Title: In silico screening and in vitro biological evaluation reveal Queuine as a promising MAP4K4 inhibitor for treating pancreatic cancer

doi: 10.1186/s12896-025-01033-w

Figure Lengend Snippet: Effects of different concentrations of Queuine and Gemcitabine on MAP4K4 activity. Absorbance values on the y-axis represent the enzyme amount measured using an ELISA kit, with different concentrations of Queuine ( A ) and Gemsitabine ( B ) indicated on the x-axis. The differences between the bar pairs marked with * ( p < 0.05) and *** ( p < 0.001) are significant

Article Snippet: The ELISA was performed following the protocol provided by the Human Mitogen-activated Protein Kinase 4 (MAP4K4) ELISA Kit, which is intended for the quantitative detection of MAP4K4 (BT Lab, Cat. no. E5689Hu) with slight modifications.

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: BMC Biotechnology

Article Title: In silico screening and in vitro biological evaluation reveal Queuine as a promising MAP4K4 inhibitor for treating pancreatic cancer

doi: 10.1186/s12896-025-01033-w

Figure Lengend Snippet: Synergetic effects of Queuine and Gemcitabine on MAP4K4 activity. Absorbance values represent the enzyme amount measured using an ELISA kit. The difference between bar pairs marked with *** are significant at p < 0.001

Article Snippet: The ELISA was performed following the protocol provided by the Human Mitogen-activated Protein Kinase 4 (MAP4K4) ELISA Kit, which is intended for the quantitative detection of MAP4K4 (BT Lab, Cat. no. E5689Hu) with slight modifications.

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay