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NCOA1 is a host factor targeted by compound 10 to regulate lipid metabolic programs (A) Validation of DARTS assay by western blot in Huh-7 cell lysates. Compd. 10 (50 μM) treatment increases the protease resistance of NCOA1 protein. (B and C) CETSA analysis reveals that compd. 10 (50 μM) induced thermal stabilization of NCOA1 is abolished by D276A and R281A mutations. Experiments were performed in HEK293T cells. (D and E) Western blot analysis shows a dose-dependent reduction of NCOA1 protein levels by compd. 10 . Data were analyzed by one-way ANOVA followed by Dunnett’s test (∗∗ p < 0.01). (F and G) CHX chase assay demonstrates accelerated degradation of NCOA1 in the presence of compd. 10 (8 μM). Student’s t test was used for statistical analysis (∗ p < 0.05, ∗∗ p < 0.01). (H and I) CHX chase analysis shows reduced stability of PPARγ protein upon compd. 10 (8 μM) treatment. Student’s t test was used for statistical analysis (∗∗ p < 0.01). (J and K) Western blot analysis shows that autophagy-lysosome inhibitors (3-MA, <t>BafA1),</t> but not the proteasome inhibitor MG132, rescue NCOA1 protein levels reduced by compd. 10 (8 μM), indicating lysosome-dependent degradation. Data were analyzed by two-way ANOVA followed by Šídák’s test (∗∗ p < 0.01, ∗∗∗ p < 0.001). Data are presented as mean ± SD from at least three independent experiments.
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PFOA/HFPO‐TA impairs PINK1 mitochondrial localization and autophagic flux in HL‐1 cells. (A) Microscopic observation was made after culture. (B) MMP and ROS levels. (C) Representative IF images of PINK1. HL‐1 cells were transfected with Mito‐DsRed. (D) Western blot analysis of p62 and LC3‐II/I protein expression under <t>Baf</t> <t>A1</t> treatment. (E) Western blot analysis of p21 and p16 protein expression under Baf A1 treatment. (F‐G) Quantitative results of proteins. (H) SA‐β‐gal staining under Baf A1 treatment. (I) Schematic diagram illustrating impaired PINK1 mitochondrial localization and mitophagic flux caused by MERCs disruption. Data are presented as the mean ± SD, n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
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NCOA1 is a host factor targeted by compound 10 to regulate lipid metabolic programs (A) Validation of DARTS assay by western blot in Huh-7 cell lysates. Compd. 10 (50 μM) treatment increases the protease resistance of NCOA1 protein. (B and C) CETSA analysis reveals that compd. 10 (50 μM) induced thermal stabilization of NCOA1 is abolished by D276A and R281A mutations. Experiments were performed in HEK293T cells. (D and E) Western blot analysis shows a dose-dependent reduction of NCOA1 protein levels by compd. 10 . Data were analyzed by one-way ANOVA followed by Dunnett’s test (∗∗ p < 0.01). (F and G) CHX chase assay demonstrates accelerated degradation of NCOA1 in the presence of compd. 10 (8 μM). Student’s t test was used for statistical analysis (∗ p < 0.05, ∗∗ p < 0.01). (H and I) CHX chase analysis shows reduced stability of PPARγ protein upon compd. 10 (8 μM) treatment. Student’s t test was used for statistical analysis (∗∗ p < 0.01). (J and K) Western blot analysis shows that autophagy-lysosome inhibitors (3-MA, BafA1), but not the proteasome inhibitor MG132, rescue NCOA1 protein levels reduced by compd. 10 (8 μM), indicating lysosome-dependent degradation. Data were analyzed by two-way ANOVA followed by Šídák’s test (∗∗ p < 0.01, ∗∗∗ p < 0.001). Data are presented as mean ± SD from at least three independent experiments.

Journal: iScience

Article Title: Targeting host lipogenesis with a diarylamide inhibitor disrupts SARS-CoV-2 replication

doi: 10.1016/j.isci.2026.117111

Figure Lengend Snippet: NCOA1 is a host factor targeted by compound 10 to regulate lipid metabolic programs (A) Validation of DARTS assay by western blot in Huh-7 cell lysates. Compd. 10 (50 μM) treatment increases the protease resistance of NCOA1 protein. (B and C) CETSA analysis reveals that compd. 10 (50 μM) induced thermal stabilization of NCOA1 is abolished by D276A and R281A mutations. Experiments were performed in HEK293T cells. (D and E) Western blot analysis shows a dose-dependent reduction of NCOA1 protein levels by compd. 10 . Data were analyzed by one-way ANOVA followed by Dunnett’s test (∗∗ p < 0.01). (F and G) CHX chase assay demonstrates accelerated degradation of NCOA1 in the presence of compd. 10 (8 μM). Student’s t test was used for statistical analysis (∗ p < 0.05, ∗∗ p < 0.01). (H and I) CHX chase analysis shows reduced stability of PPARγ protein upon compd. 10 (8 μM) treatment. Student’s t test was used for statistical analysis (∗∗ p < 0.01). (J and K) Western blot analysis shows that autophagy-lysosome inhibitors (3-MA, BafA1), but not the proteasome inhibitor MG132, rescue NCOA1 protein levels reduced by compd. 10 (8 μM), indicating lysosome-dependent degradation. Data were analyzed by two-way ANOVA followed by Šídák’s test (∗∗ p < 0.01, ∗∗∗ p < 0.001). Data are presented as mean ± SD from at least three independent experiments.

Article Snippet: Bafilomycin A1 (BafA1) , MCE , Cat# HY-100558.

Techniques: Biomarker Discovery, Western Blot

PFOA/HFPO‐TA impairs PINK1 mitochondrial localization and autophagic flux in HL‐1 cells. (A) Microscopic observation was made after culture. (B) MMP and ROS levels. (C) Representative IF images of PINK1. HL‐1 cells were transfected with Mito‐DsRed. (D) Western blot analysis of p62 and LC3‐II/I protein expression under Baf A1 treatment. (E) Western blot analysis of p21 and p16 protein expression under Baf A1 treatment. (F‐G) Quantitative results of proteins. (H) SA‐β‐gal staining under Baf A1 treatment. (I) Schematic diagram illustrating impaired PINK1 mitochondrial localization and mitophagic flux caused by MERCs disruption. Data are presented as the mean ± SD, n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Journal: Advanced Science

Article Title: CDK4/MERCs/PINK1 Axis Drives PFOA/HFPO‐TA‐Induced Cardiac Senescence via Mitophagy Defect and cGAS‐STING Activation: In Vitro Amelioration by Cycloastragenol

doi: 10.1002/advs.77280

Figure Lengend Snippet: PFOA/HFPO‐TA impairs PINK1 mitochondrial localization and autophagic flux in HL‐1 cells. (A) Microscopic observation was made after culture. (B) MMP and ROS levels. (C) Representative IF images of PINK1. HL‐1 cells were transfected with Mito‐DsRed. (D) Western blot analysis of p62 and LC3‐II/I protein expression under Baf A1 treatment. (E) Western blot analysis of p21 and p16 protein expression under Baf A1 treatment. (F‐G) Quantitative results of proteins. (H) SA‐β‐gal staining under Baf A1 treatment. (I) Schematic diagram illustrating impaired PINK1 mitochondrial localization and mitophagic flux caused by MERCs disruption. Data are presented as the mean ± SD, n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: For cellular stimulation, HL‐1 cells were treated with different reagents, including 20 nM Baf A1 and 1 μM H‐151 (MCE, USA).

Techniques: Transfection, Western Blot, Expressing, Staining, Disruption