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: 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: 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