Journal: Antioxidants (Basel, Switzerland)

Article Title: Aster-B Modulates Oxidative Stress Responses and Carotenoid Distribution in ARPE-19 Cells.

doi: 10.3390/antiox14050575

Figure Lengend Snippet: Figure 1. CRISPR/dCas9 activation of endogenous GRAMD1B gene expression in ARPE19 GRAMD1B-null cells. (A) ARPE-19 cells were transfected with scrambled DNA at 0.5, 1, and 1.5 µg DNA per well and GRAMD1B plasmid DNA at 0.5, 1, and 1.5 µg DNA per well, respectively. Western blot analysis was conducted with 50 µg of total protein per lane and showed that GRAMD1B is expressed before clonal selection and becomes enriched in post-clonal ARPE-19 cells (25 and 50 µg per well) treated with GRAMD1B plasmid DNA (1.5 µg per well). In contrast, GRAMD1B expression is absent in control cells treated with scrambled control DNA (1.5 µg per well). A549 cells were used as a positive control for GRAMD1B expression. β-Actin was used as a loading control. (B) Confocal imaging analysis with ARPE-19 cells transfected with plasmid DNA confirmed that GRAMD1B protein is expressed and mostly localized in the cytoplasm. Similar results were obtained from WB and ICC in more than three independent experiments. The scale bar is 10 µm.

Article Snippet: The antibodies used included GRAMD1B (Proteintech), COXIV, Caspase-3, PARP, and Histone H3 (Cell Signaling Technology) at a dilution of 1:1000.

Techniques: CRISPR, Activation Assay, Gene Expression, Transfection, Plasmid Preparation, Western Blot, Selection, Expressing, Control, Positive Control, Imaging

Journal: Antioxidants

Article Title: Aster-B Modulates Oxidative Stress Responses and Carotenoid Distribution in ARPE-19 Cells

doi: 10.3390/antiox14050575

Figure Lengend Snippet: CRISPR/dCas9 activation of endogenous GRAMD1B gene expression in ARPE19 GRAMD1B -null cells. ( A ) ARPE-19 cells were transfected with scrambled DNA at 0.5, 1, and 1.5 μg DNA per well and GRAMD1B plasmid DNA at 0.5, 1, and 1.5 μg DNA per well, respectively. Western blot analysis was conducted with 50 μg of total protein per lane and showed that GRAMD1B is expressed before clonal selection and becomes enriched in post-clonal ARPE-19 cells (25 and 50 μg per well) treated with GRAMD1B plasmid DNA (1.5 μg per well). In contrast, GRAMD1B expression is absent in control cells treated with scrambled control DNA (1.5 μg per well). A549 cells were used as a positive control for GRAMD1B expression. β-Actin was used as a loading control. ( B ) Confocal imaging analysis with ARPE-19 cells transfected with plasmid DNA confirmed that GRAMD1B protein is expressed and mostly localized in the cytoplasm. Similar results were obtained from WB and ICC in more than three independent experiments. The scale bar is 10 μm.

Article Snippet: Control CRISPR Activation Plasmid (sc-437275), GRAMD1B CRISPR Activation Plasmid (h) (sc-408929-ACT), plasmid transfection medium (sc-108062), Ultra Cruz transfection reagent (sc-395739), hygromycin B (sc-29067), puromycin (sc-108071), and blasticidin S HCl (sc-204655A) (Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: CRISPR, Activation Assay, Gene Expression, Transfection, Plasmid Preparation, Western Blot, Selection, Expressing, Control, Positive Control, Imaging

Journal: Molecular Metabolism

Article Title: Aster-B coordinates with Arf1 to regulate mitochondrial cholesterol transport

doi: 10.1016/j.molmet.2020.101055

Figure Lengend Snippet: Ablation of Aster-B impairs uptake of cholesterol and cholesterol esters. (A) Time-lapse confocal imaging analysis depicting the cholesterol uptake in C2C12 vector control (VC) and Aster-B knockout (KO) cells. Cells were depleted of endogenous cholesterol using 0.5% methyl-β-cyclodextrin treatment for 2 hr and then treated with 50 μM of 25-NBD-Cholesterol for the times indicated. Nuclei are stained with Hoechst 33342. (B) Time-lapse confocal imaging analysis depicting the cholesterol ester uptake in C2C12 VC and Aster-B KO cells. Cells were starved in KRPH buffer for 30 min and treated with 10 μM of CholEsteryl BODIPY™ 542/563 C11 for the times indicated. Nuclei are stained with Hoechst 33342. (C) Fluorescence density analysis of green fluorescent cholesterol treated VC and KO cells at 30 minutes, n=10. (D) Fluorescence density analysis of CholEsteryl BODIPY™ 542/563 C11-treated VC and KO cells at 12 min n = 10. ∗∗∗∗p < 0.0001 by student's t-test.

Article Snippet: For immunoblotting, C2C12 Vector Control, Aster-B Knockout cells, and cells transfected with a WT Aster-B expressing plasmid (RG219269) from Origene or Mutant Plasmid Δ2-31 were depleted and re-stimulated with cholesterol in the same manner as described above.

Techniques: Imaging, Plasmid Preparation, Knock-Out, Staining, Fluorescence

Journal: Molecular Metabolism

Article Title: Aster-B coordinates with Arf1 to regulate mitochondrial cholesterol transport

doi: 10.1016/j.molmet.2020.101055

Figure Lengend Snippet: Aster-B deficiency impairs the transport of cholesterol and fatty acids derived from cholesterol esters into mitochondria. (A) Confocal imaging analysis depicting the mitochondrial transport of cholesterol ester in vector control (VC) and Aster-B knockout (KO) cells. Cells were starved in KRPH buffer for 30 min and then treated with 10 μM of CholEsteryl BODIPY™ 542/563 C11 for 10 min. Digitonin-treated cells were starved in KRPH buffer for 20 min and then pre-treated with 10 μM of digitonin for 10 min, washed twice in KRPH buffer, and then treated with 10 μM of CholEsteryl BODIPY™ 542/563 C11 for 10 min. Mitochondria and nuclei were stained by Mitotracker-Green and Hoechst 33342, respectively. (B) Pearson's Co-localization Coefficient of Mitotracker Green and CholEsteryl BODIPY™ 542/563 C11 in the VC and KO cells. n = 20. ∗∗∗∗p < 0.0001 by student's t-test. (C) Pearson's Co-localization Coefficient of Mitotracker Green and CholEsteryl BODIPY™ 542/563 C11 in vehicle and digitonin-treated Aster-B KO cells. n = 20. (D) Confocal images of C2C12 cells pre-treated with vehicle or 300 nM of etomoxir for 6 h. Cells were then starved for 30 min and treated with CholEsteryl BODIPY™ 542/563 C11 for 10 min. Mitochondria were stained with Mitotracker-Green. (E) Pearson's Co-localization Coefficient of Mitotracker-Green and CholEsteryl BODIPY™ 542/563 C11 in D. n = 20. ∗∗∗∗p < 0.0001 by student's t-test. (F) Confocal imaging analysis depicting the co-localization of cholesterol with mitochondria in VC and Aster-B KO cells. Cells were treated with MCD, permeabilized by digitonin, and then incubated with 16:0 TopFluor® cholesterol. Mitochondria were stained with Mitotracker-Red. (G) Pearson's Co-localization Coefficient of Mitotracker-Red and 16:0 TopFluor® cholesterol in F. n = 20. ∗∗∗p < 0.001 by student's t-test.

Article Snippet: For immunoblotting, C2C12 Vector Control, Aster-B Knockout cells, and cells transfected with a WT Aster-B expressing plasmid (RG219269) from Origene or Mutant Plasmid Δ2-31 were depleted and re-stimulated with cholesterol in the same manner as described above.

Techniques: Derivative Assay, Imaging, Plasmid Preparation, Knock-Out, Staining, Incubation

Journal: Molecular Metabolism

Article Title: Aster-B coordinates with Arf1 to regulate mitochondrial cholesterol transport

doi: 10.1016/j.molmet.2020.101055

Figure Lengend Snippet: MTS is required for the tethering of Aster-B at ER and mitochondrial contact sites. (A) Schematic representation of Aster-B protein showing deleted region (2–31) of plasmid with MTS highlighted in yellow. (B–C) Confocal images of C2C12 Aster-B knockout (KO)cells transfected with GFP labeled Aster-B or Δ2-31-Aster-B and an ER localized DsRed-ER protein (B) or a mitochondria resident protein, Mito-DsRed (C), respectively. Cells were starved for 30 min in KRPH buffer followed by 10 min of 10 μM of CholEsteryl BODIPY™ 542/563 C11 stimulation. (D–E) Subcellular fractionation and Western blot analysis of 293A cells transfected with either wild-type (WT) or Δ2-31 Aster-B plasmids. Cells were starved for 30 min in KRPH buffer and then treated with 10 μM of non-fluorescent cholesterol esters for 10 min. Micro, microsome; Cr. Mito, crude mitochondria, pure mito, pure mitochondria; cyto, cytosol. (F) Densitometric analysis showing the fold difference of WT Aster-B to Δ2-31 in the MAM, using Bip as an internal control, n = 3. ∗∗∗∗p < 0.0001 by two-way ANOVA. (G) Densitometric analysis showing the fold difference of WT Aster-B to Δ2-31 in the purified mitochondrial fraction using Tom20 as an internal control n = 3. ∗∗∗p < 0.001 by two-way ANOVA.

Article Snippet: For immunoblotting, C2C12 Vector Control, Aster-B Knockout cells, and cells transfected with a WT Aster-B expressing plasmid (RG219269) from Origene or Mutant Plasmid Δ2-31 were depleted and re-stimulated with cholesterol in the same manner as described above.

Techniques: Plasmid Preparation, Knock-Out, Transfection, Labeling, Fractionation, Western Blot, Purification

Journal: Molecular Metabolism

Article Title: Aster-B coordinates with Arf1 to regulate mitochondrial cholesterol transport

doi: 10.1016/j.molmet.2020.101055

Figure Lengend Snippet: Deletion of MTS of Aster-B impairs mitochondrial cholesterol trafficking. (A–B) Confocal imaging analysis depicting the mitochondrial cholesterol transport in C2C12 Aster-B knockout (KO) cells re-expressing GFP-Aster-B WT (A) or Δ2-31 mutant (B). Mitochondria were labeled by transfecting cells with mito-BFP. Cells were starved for 20 min in KRPH buffer, followed by 10 min of permeabilization with 10 μM of digitonin, and then treated with 10 μM of CholEsteryl BODIPY™ 542/563 C11 for 10 min. (C–E) Total cholesterol level (C) and cholesterol levels in pure mitochondria (D) and MAM (E) in Aster-B KO cells re-expressing GFP-Aster-B WT or Δ2-31 mutant. Cells were treated with MCD (-CHOL) or permeabilized by digitonin after MCD treatment and re-incubated with 50 μM of cholesterol (+CHOL). n = 3. ∗∗p < 0.01, ∗∗∗p < 0.001 by two -ANOVA.

Article Snippet: For immunoblotting, C2C12 Vector Control, Aster-B Knockout cells, and cells transfected with a WT Aster-B expressing plasmid (RG219269) from Origene or Mutant Plasmid Δ2-31 were depleted and re-stimulated with cholesterol in the same manner as described above.

Techniques: Imaging, Knock-Out, Expressing, Mutagenesis, Labeling, Incubation

Journal: Molecular Metabolism

Article Title: Aster-B coordinates with Arf1 to regulate mitochondrial cholesterol transport

doi: 10.1016/j.molmet.2020.101055

Figure Lengend Snippet: Arf1 is required for cholesterol transport into mitochondria. (A) Confocal imaging analysis depicting the mitochondrial cholesterol transport in HeLa vector control (VC) and Arf1 knockout (Arf1-KO) cells. Cells were starved in KRPH buffer for 30 min and then treated with 10 μM of CholEsteryl BODIPY™ 542/563 C11 for 10 min. (B) Confocal imaging analysis depicting the mitochondrial cholesterol transport in C2C12 cells treated with 10 μM of Exo-2 for 2 h followed by incubation with 10 μM of CholEsteryl BODIPY™ 542/563 C11 for 10 min. Nuclei were stained with Hoechst 33342, and mitochondria were stained with Mitotracker-Green. (C–E) Total cholesterol level (C) and cholesterol levels in pure mitochondria (D) and MAM (E) in HeLa VC and Arf1 KO cells. Cells were treated with MCD (-CHOL), or pemeabilized by digitonin after MCD treatment and re-incubated with 50 μM of cholesterol (+CHOL). n = 3. ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 by two-way ANOVA.

Article Snippet: For immunoblotting, C2C12 Vector Control, Aster-B Knockout cells, and cells transfected with a WT Aster-B expressing plasmid (RG219269) from Origene or Mutant Plasmid Δ2-31 were depleted and re-stimulated with cholesterol in the same manner as described above.

Techniques: Imaging, Plasmid Preparation, Knock-Out, Incubation, Staining

Journal: Molecular Metabolism

Article Title: Aster-B coordinates with Arf1 to regulate mitochondrial cholesterol transport

doi: 10.1016/j.molmet.2020.101055

Figure Lengend Snippet: Ablation of Aster-B or inhibition of Arf1 causes mitochondrial dysfunction. (A) Seahorse XF analysis showing oxygen consumption rate (OCR) of C2C12 vector control with vehicle (VC) or 10 μM of Exo-2 (VC Exo-2) and Aster-B knockout (KO) cells treated with MCD for 2 hr and then treated with 25 μM non-fluorescent cholesterol ester for 1 h. Cells were treated with vehicle or 10 μM of Exo-2 throughout the assay. (B) Basal, (C) maximum, (D) ATP-linked respiration of the Seahorse XF analysis is shown. n = 5. ∗∗p < 0.01, ∗∗∗∗p < 0.0001 by student's t-test.

Article Snippet: For immunoblotting, C2C12 Vector Control, Aster-B Knockout cells, and cells transfected with a WT Aster-B expressing plasmid (RG219269) from Origene or Mutant Plasmid Δ2-31 were depleted and re-stimulated with cholesterol in the same manner as described above.

Techniques: Inhibition, Plasmid Preparation, Knock-Out