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Induction of ferroptosis-like phenotype by JIB-04 drives synergism with VEN. (A-C) OCI-AML3 and MOLM-1 cells were treated with 40nM/50nM JIB-04, 8mM/4mM NAC <t>(antioxidant),</t> <t>25μM</t> <t>Z-VAD-FMK</t> <t>(caspase</t> inhibitor), or the combination JIB-04+NAC and JIB-04+Z-VAD-FMK for up to 48 hours (mean ± SD; n = 3). Cells were stained for ferroptosis markers and analyzed by flow cytometry. JIB-04 treatment group was normalized to the DMSO group. Statistical significance was calculated by 2-way ANOVA with Tukey multiple-comparison test. (A) MitoSOX red for mitochondrial ROS at 24 hours. (B) MitoPerOx for mitochondrial lipid peroxidation at 24 hours. (C) BODIPY C11 for lipid peroxidation at 48 hours. (D) HMOX1 protein levels were visualized by immunoblotting in AML cell lines treated with DMSO, JIB-04, or JIB-04+NAC for 24 hours. (E-F) Quantification of apoptosis by annexin V/PI staining in OCI-AML3 and MOLM-13 cells after treatment with 40nM/50nM JIB-04, 1μM/1nM VEN, 8mM/4mM NAC, or the indicated drug combinations. Statistical significance was calculated by ordinary 1-way ANOVA with Tukey multiple-comparison test. ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001; ∗∗∗∗ P < .0001. Z-VAD, Z-VAD-FMK.
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Induction of ferroptosis-like phenotype by JIB-04 drives synergism with VEN. (A-C) OCI-AML3 and MOLM-1 cells were treated with 40nM/50nM JIB-04, 8mM/4mM NAC <t>(antioxidant),</t> <t>25μM</t> <t>Z-VAD-FMK</t> <t>(caspase</t> inhibitor), or the combination JIB-04+NAC and JIB-04+Z-VAD-FMK for up to 48 hours (mean ± SD; n = 3). Cells were stained for ferroptosis markers and analyzed by flow cytometry. JIB-04 treatment group was normalized to the DMSO group. Statistical significance was calculated by 2-way ANOVA with Tukey multiple-comparison test. (A) MitoSOX red for mitochondrial ROS at 24 hours. (B) MitoPerOx for mitochondrial lipid peroxidation at 24 hours. (C) BODIPY C11 for lipid peroxidation at 48 hours. (D) HMOX1 protein levels were visualized by immunoblotting in AML cell lines treated with DMSO, JIB-04, or JIB-04+NAC for 24 hours. (E-F) Quantification of apoptosis by annexin V/PI staining in OCI-AML3 and MOLM-13 cells after treatment with 40nM/50nM JIB-04, 1μM/1nM VEN, 8mM/4mM NAC, or the indicated drug combinations. Statistical significance was calculated by ordinary 1-way ANOVA with Tukey multiple-comparison test. ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001; ∗∗∗∗ P < .0001. Z-VAD, Z-VAD-FMK.
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Induction of ferroptosis-like phenotype by JIB-04 drives synergism with VEN. (A-C) OCI-AML3 and MOLM-1 cells were treated with 40nM/50nM JIB-04, 8mM/4mM NAC <t>(antioxidant),</t> <t>25μM</t> <t>Z-VAD-FMK</t> <t>(caspase</t> inhibitor), or the combination JIB-04+NAC and JIB-04+Z-VAD-FMK for up to 48 hours (mean ± SD; n = 3). Cells were stained for ferroptosis markers and analyzed by flow cytometry. JIB-04 treatment group was normalized to the DMSO group. Statistical significance was calculated by 2-way ANOVA with Tukey multiple-comparison test. (A) MitoSOX red for mitochondrial ROS at 24 hours. (B) MitoPerOx for mitochondrial lipid peroxidation at 24 hours. (C) BODIPY C11 for lipid peroxidation at 48 hours. (D) HMOX1 protein levels were visualized by immunoblotting in AML cell lines treated with DMSO, JIB-04, or JIB-04+NAC for 24 hours. (E-F) Quantification of apoptosis by annexin V/PI staining in OCI-AML3 and MOLM-13 cells after treatment with 40nM/50nM JIB-04, 1μM/1nM VEN, 8mM/4mM NAC, or the indicated drug combinations. Statistical significance was calculated by ordinary 1-way ANOVA with Tukey multiple-comparison test. ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001; ∗∗∗∗ P < .0001. Z-VAD, Z-VAD-FMK.
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Induction of ferroptosis-like phenotype by JIB-04 drives synergism with VEN. (A-C) OCI-AML3 and MOLM-1 cells were treated with 40nM/50nM JIB-04, 8mM/4mM NAC <t>(antioxidant),</t> <t>25μM</t> <t>Z-VAD-FMK</t> <t>(caspase</t> inhibitor), or the combination JIB-04+NAC and JIB-04+Z-VAD-FMK for up to 48 hours (mean ± SD; n = 3). Cells were stained for ferroptosis markers and analyzed by flow cytometry. JIB-04 treatment group was normalized to the DMSO group. Statistical significance was calculated by 2-way ANOVA with Tukey multiple-comparison test. (A) MitoSOX red for mitochondrial ROS at 24 hours. (B) MitoPerOx for mitochondrial lipid peroxidation at 24 hours. (C) BODIPY C11 for lipid peroxidation at 48 hours. (D) HMOX1 protein levels were visualized by immunoblotting in AML cell lines treated with DMSO, JIB-04, or JIB-04+NAC for 24 hours. (E-F) Quantification of apoptosis by annexin V/PI staining in OCI-AML3 and MOLM-13 cells after treatment with 40nM/50nM JIB-04, 1μM/1nM VEN, 8mM/4mM NAC, or the indicated drug combinations. Statistical significance was calculated by ordinary 1-way ANOVA with Tukey multiple-comparison test. ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001; ∗∗∗∗ P < .0001. Z-VAD, Z-VAD-FMK.
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Induction of ferroptosis-like phenotype by JIB-04 drives synergism with VEN. (A-C) OCI-AML3 and MOLM-1 cells were treated with 40nM/50nM JIB-04, 8mM/4mM NAC (antioxidant), 25μM Z-VAD-FMK (caspase inhibitor), or the combination JIB-04+NAC and JIB-04+Z-VAD-FMK for up to 48 hours (mean ± SD; n = 3). Cells were stained for ferroptosis markers and analyzed by flow cytometry. JIB-04 treatment group was normalized to the DMSO group. Statistical significance was calculated by 2-way ANOVA with Tukey multiple-comparison test. (A) MitoSOX red for mitochondrial ROS at 24 hours. (B) MitoPerOx for mitochondrial lipid peroxidation at 24 hours. (C) BODIPY C11 for lipid peroxidation at 48 hours. (D) HMOX1 protein levels were visualized by immunoblotting in AML cell lines treated with DMSO, JIB-04, or JIB-04+NAC for 24 hours. (E-F) Quantification of apoptosis by annexin V/PI staining in OCI-AML3 and MOLM-13 cells after treatment with 40nM/50nM JIB-04, 1μM/1nM VEN, 8mM/4mM NAC, or the indicated drug combinations. Statistical significance was calculated by ordinary 1-way ANOVA with Tukey multiple-comparison test. ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001; ∗∗∗∗ P < .0001. Z-VAD, Z-VAD-FMK.

Journal: Blood Neoplasia

Article Title: The KDM-family inhibitor JIB-04 sensitizes AML cells to venetoclax by inducing a ferroptosis-like phenotype

doi: 10.1016/j.bneo.2026.100236

Figure Lengend Snippet: Induction of ferroptosis-like phenotype by JIB-04 drives synergism with VEN. (A-C) OCI-AML3 and MOLM-1 cells were treated with 40nM/50nM JIB-04, 8mM/4mM NAC (antioxidant), 25μM Z-VAD-FMK (caspase inhibitor), or the combination JIB-04+NAC and JIB-04+Z-VAD-FMK for up to 48 hours (mean ± SD; n = 3). Cells were stained for ferroptosis markers and analyzed by flow cytometry. JIB-04 treatment group was normalized to the DMSO group. Statistical significance was calculated by 2-way ANOVA with Tukey multiple-comparison test. (A) MitoSOX red for mitochondrial ROS at 24 hours. (B) MitoPerOx for mitochondrial lipid peroxidation at 24 hours. (C) BODIPY C11 for lipid peroxidation at 48 hours. (D) HMOX1 protein levels were visualized by immunoblotting in AML cell lines treated with DMSO, JIB-04, or JIB-04+NAC for 24 hours. (E-F) Quantification of apoptosis by annexin V/PI staining in OCI-AML3 and MOLM-13 cells after treatment with 40nM/50nM JIB-04, 1μM/1nM VEN, 8mM/4mM NAC, or the indicated drug combinations. Statistical significance was calculated by ordinary 1-way ANOVA with Tukey multiple-comparison test. ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001; ∗∗∗∗ P < .0001. Z-VAD, Z-VAD-FMK.

Article Snippet: AML cells were treated with JIB-04, the antioxidant NAC, the pan caspase inhibitor Z-VAD-FMK (MedChemExpress), or indicated combinations for up to 48 hours.

Techniques: Staining, Flow Cytometry, Comparison, Western Blot