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a C273 increases resistance to oxidative stress induced by H 2 O 2 in MC65 Tet-Off cells expressing Aβ. Cells were pretreated with C273 prior to H 2 O 2 exposure, and cell viability was assessed to determine LD 50 values. LD 50 values were calculated by nonlinear regression using GraphPad Prism. b, c C273 restores intracellular NADPH ( b ) and GSH ( c ) levels in MC65 Tet-Off cells. d C273 suppresses IL-1β-induced NF-κB activation. NF-κB reporter <t>HEK293</t> cells were pretreated for 24 h with C273 (50 nM, 500 nM, or 5 μM), AICAR (0.5 mM), or metformin (0.5 mM), followed by stimulation with IL-1β (1 ng/ml) for 6 h. NF-κB luciferase activity was measured using the Luciferase Assay System (Promega, E1501). n = 4 independent experiments. e C273 restores mitochondrial biogenesis, expression of ETC complexes, autophagy markers, and antioxidant proteins in MC65 Tet-Off cells. MC65 cells were cultured under Tet-On (Aβ-) or Tet-Off (Aβ+) conditions and treated with vehicle or C273 (5 nM, 50 nM, or 500 nM) for 24 h. Protein levels were analyzed by Western blot. Statistical analysis was performed via one-way ANOVA to compare the vehicle-, metformin-, AICAR- and C273-treated groups. Data are presented as mean ± SD. * P < 0.05; ** P < 0.01.
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a C273 increases resistance to oxidative stress induced by H 2 O 2 in MC65 Tet-Off cells expressing Aβ. Cells were pretreated with C273 prior to H 2 O 2 exposure, and cell viability was assessed to determine LD 50 values. LD 50 values were calculated by nonlinear regression using GraphPad Prism. b, c C273 restores intracellular NADPH ( b ) and GSH ( c ) levels in MC65 Tet-Off cells. d C273 suppresses IL-1β-induced NF-κB activation. NF-κB reporter <t>HEK293</t> cells were pretreated for 24 h with C273 (50 nM, 500 nM, or 5 μM), AICAR (0.5 mM), or metformin (0.5 mM), followed by stimulation with IL-1β (1 ng/ml) for 6 h. NF-κB luciferase activity was measured using the Luciferase Assay System (Promega, E1501). n = 4 independent experiments. e C273 restores mitochondrial biogenesis, expression of ETC complexes, autophagy markers, and antioxidant proteins in MC65 Tet-Off cells. MC65 cells were cultured under Tet-On (Aβ-) or Tet-Off (Aβ+) conditions and treated with vehicle or C273 (5 nM, 50 nM, or 500 nM) for 24 h. Protein levels were analyzed by Western blot. Statistical analysis was performed via one-way ANOVA to compare the vehicle-, metformin-, AICAR- and C273-treated groups. Data are presented as mean ± SD. * P < 0.05; ** P < 0.01.
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a C273 increases resistance to oxidative stress induced by H 2 O 2 in MC65 Tet-Off cells expressing Aβ. Cells were pretreated with C273 prior to H 2 O 2 exposure, and cell viability was assessed to determine LD 50 values. LD 50 values were calculated by nonlinear regression using GraphPad Prism. b, c C273 restores intracellular NADPH ( b ) and GSH ( c ) levels in MC65 Tet-Off cells. d C273 suppresses IL-1β-induced NF-κB activation. NF-κB reporter <t>HEK293</t> cells were pretreated for 24 h with C273 (50 nM, 500 nM, or 5 μM), AICAR (0.5 mM), or metformin (0.5 mM), followed by stimulation with IL-1β (1 ng/ml) for 6 h. NF-κB luciferase activity was measured using the Luciferase Assay System (Promega, E1501). n = 4 independent experiments. e C273 restores mitochondrial biogenesis, expression of ETC complexes, autophagy markers, and antioxidant proteins in MC65 Tet-Off cells. MC65 cells were cultured under Tet-On (Aβ-) or Tet-Off (Aβ+) conditions and treated with vehicle or C273 (5 nM, 50 nM, or 500 nM) for 24 h. Protein levels were analyzed by Western blot. Statistical analysis was performed via one-way ANOVA to compare the vehicle-, metformin-, AICAR- and C273-treated groups. Data are presented as mean ± SD. * P < 0.05; ** P < 0.01.
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a C273 increases resistance to oxidative stress induced by H 2 O 2 in MC65 Tet-Off cells expressing Aβ. Cells were pretreated with C273 prior to H 2 O 2 exposure, and cell viability was assessed to determine LD 50 values. LD 50 values were calculated by nonlinear regression using GraphPad Prism. b, c C273 restores intracellular NADPH ( b ) and GSH ( c ) levels in MC65 Tet-Off cells. d C273 suppresses IL-1β-induced NF-κB activation. NF-κB reporter <t>HEK293</t> cells were pretreated for 24 h with C273 (50 nM, 500 nM, or 5 μM), AICAR (0.5 mM), or metformin (0.5 mM), followed by stimulation with IL-1β (1 ng/ml) for 6 h. NF-κB luciferase activity was measured using the Luciferase Assay System (Promega, E1501). n = 4 independent experiments. e C273 restores mitochondrial biogenesis, expression of ETC complexes, autophagy markers, and antioxidant proteins in MC65 Tet-Off cells. MC65 cells were cultured under Tet-On (Aβ-) or Tet-Off (Aβ+) conditions and treated with vehicle or C273 (5 nM, 50 nM, or 500 nM) for 24 h. Protein levels were analyzed by Western blot. Statistical analysis was performed via one-way ANOVA to compare the vehicle-, metformin-, AICAR- and C273-treated groups. Data are presented as mean ± SD. * P < 0.05; ** P < 0.01.
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a C273 increases resistance to oxidative stress induced by H 2 O 2 in MC65 Tet-Off cells expressing Aβ. Cells were pretreated with C273 prior to H 2 O 2 exposure, and cell viability was assessed to determine LD 50 values. LD 50 values were calculated by nonlinear regression using GraphPad Prism. b, c C273 restores intracellular NADPH ( b ) and GSH ( c ) levels in MC65 Tet-Off cells. d C273 suppresses IL-1β-induced NF-κB activation. NF-κB reporter <t>HEK293</t> cells were pretreated for 24 h with C273 (50 nM, 500 nM, or 5 μM), AICAR (0.5 mM), or metformin (0.5 mM), followed by stimulation with IL-1β (1 ng/ml) for 6 h. NF-κB luciferase activity was measured using the Luciferase Assay System (Promega, E1501). n = 4 independent experiments. e C273 restores mitochondrial biogenesis, expression of ETC complexes, autophagy markers, and antioxidant proteins in MC65 Tet-Off cells. MC65 cells were cultured under Tet-On (Aβ-) or Tet-Off (Aβ+) conditions and treated with vehicle or C273 (5 nM, 50 nM, or 500 nM) for 24 h. Protein levels were analyzed by Western blot. Statistical analysis was performed via one-way ANOVA to compare the vehicle-, metformin-, AICAR- and C273-treated groups. Data are presented as mean ± SD. * P < 0.05; ** P < 0.01.
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a C273 increases resistance to oxidative stress induced by H 2 O 2 in MC65 Tet-Off cells expressing Aβ. Cells were pretreated with C273 prior to H 2 O 2 exposure, and cell viability was assessed to determine LD 50 values. LD 50 values were calculated by nonlinear regression using GraphPad Prism. b, c C273 restores intracellular NADPH ( b ) and GSH ( c ) levels in MC65 Tet-Off cells. d C273 suppresses IL-1β-induced NF-κB activation. NF-κB reporter <t>HEK293</t> cells were pretreated for 24 h with C273 (50 nM, 500 nM, or 5 μM), AICAR (0.5 mM), or metformin (0.5 mM), followed by stimulation with IL-1β (1 ng/ml) for 6 h. NF-κB luciferase activity was measured using the Luciferase Assay System (Promega, E1501). n = 4 independent experiments. e C273 restores mitochondrial biogenesis, expression of ETC complexes, autophagy markers, and antioxidant proteins in MC65 Tet-Off cells. MC65 cells were cultured under Tet-On (Aβ-) or Tet-Off (Aβ+) conditions and treated with vehicle or C273 (5 nM, 50 nM, or 500 nM) for 24 h. Protein levels were analyzed by Western blot. Statistical analysis was performed via one-way ANOVA to compare the vehicle-, metformin-, AICAR- and C273-treated groups. Data are presented as mean ± SD. * P < 0.05; ** P < 0.01.
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a C273 increases resistance to oxidative stress induced by H 2 O 2 in MC65 Tet-Off cells expressing Aβ. Cells were pretreated with C273 prior to H 2 O 2 exposure, and cell viability was assessed to determine LD 50 values. LD 50 values were calculated by nonlinear regression using GraphPad Prism. b, c C273 restores intracellular NADPH ( b ) and GSH ( c ) levels in MC65 Tet-Off cells. d C273 suppresses IL-1β-induced NF-κB activation. NF-κB reporter <t>HEK293</t> cells were pretreated for 24 h with C273 (50 nM, 500 nM, or 5 μM), AICAR (0.5 mM), or metformin (0.5 mM), followed by stimulation with IL-1β (1 ng/ml) for 6 h. NF-κB luciferase activity was measured using the Luciferase Assay System (Promega, E1501). n = 4 independent experiments. e C273 restores mitochondrial biogenesis, expression of ETC complexes, autophagy markers, and antioxidant proteins in MC65 Tet-Off cells. MC65 cells were cultured under Tet-On (Aβ-) or Tet-Off (Aβ+) conditions and treated with vehicle or C273 (5 nM, 50 nM, or 500 nM) for 24 h. Protein levels were analyzed by Western blot. Statistical analysis was performed via one-way ANOVA to compare the vehicle-, metformin-, AICAR- and C273-treated groups. Data are presented as mean ± SD. * P < 0.05; ** P < 0.01.
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a C273 increases resistance to oxidative stress induced by H 2 O 2 in MC65 Tet-Off cells expressing Aβ. Cells were pretreated with C273 prior to H 2 O 2 exposure, and cell viability was assessed to determine LD 50 values. LD 50 values were calculated by nonlinear regression using GraphPad Prism. b, c C273 restores intracellular NADPH ( b ) and GSH ( c ) levels in MC65 Tet-Off cells. d C273 suppresses IL-1β-induced NF-κB activation. NF-κB reporter HEK293 cells were pretreated for 24 h with C273 (50 nM, 500 nM, or 5 μM), AICAR (0.5 mM), or metformin (0.5 mM), followed by stimulation with IL-1β (1 ng/ml) for 6 h. NF-κB luciferase activity was measured using the Luciferase Assay System (Promega, E1501). n = 4 independent experiments. e C273 restores mitochondrial biogenesis, expression of ETC complexes, autophagy markers, and antioxidant proteins in MC65 Tet-Off cells. MC65 cells were cultured under Tet-On (Aβ-) or Tet-Off (Aβ+) conditions and treated with vehicle or C273 (5 nM, 50 nM, or 500 nM) for 24 h. Protein levels were analyzed by Western blot. Statistical analysis was performed via one-way ANOVA to compare the vehicle-, metformin-, AICAR- and C273-treated groups. Data are presented as mean ± SD. * P < 0.05; ** P < 0.01.

Journal: bioRxiv

Article Title: Discovery and Preclinical Validation of a Clinically Optimized Mitochondrial Complex I Modulator for Alzheimer’s Disease

doi: 10.64898/2026.04.10.717554

Figure Lengend Snippet: a C273 increases resistance to oxidative stress induced by H 2 O 2 in MC65 Tet-Off cells expressing Aβ. Cells were pretreated with C273 prior to H 2 O 2 exposure, and cell viability was assessed to determine LD 50 values. LD 50 values were calculated by nonlinear regression using GraphPad Prism. b, c C273 restores intracellular NADPH ( b ) and GSH ( c ) levels in MC65 Tet-Off cells. d C273 suppresses IL-1β-induced NF-κB activation. NF-κB reporter HEK293 cells were pretreated for 24 h with C273 (50 nM, 500 nM, or 5 μM), AICAR (0.5 mM), or metformin (0.5 mM), followed by stimulation with IL-1β (1 ng/ml) for 6 h. NF-κB luciferase activity was measured using the Luciferase Assay System (Promega, E1501). n = 4 independent experiments. e C273 restores mitochondrial biogenesis, expression of ETC complexes, autophagy markers, and antioxidant proteins in MC65 Tet-Off cells. MC65 cells were cultured under Tet-On (Aβ-) or Tet-Off (Aβ+) conditions and treated with vehicle or C273 (5 nM, 50 nM, or 500 nM) for 24 h. Protein levels were analyzed by Western blot. Statistical analysis was performed via one-way ANOVA to compare the vehicle-, metformin-, AICAR- and C273-treated groups. Data are presented as mean ± SD. * P < 0.05; ** P < 0.01.

Article Snippet: NF-κB Reporter (Luc) HEK293 cell line and the antioxidant response element (ARE) Luciferase Reporter HepG2 hepatic cell line were purchased from BPS Bioscience (CA, USA) and were cultured in accordance of manufacturer instructions.

Techniques: Expressing, Activation Assay, Luciferase, Activity Assay, Cell Culture, Western Blot