Journal: PLoS Biology

Article Title: Copper-dependent amino oxidase 3 governs selection of metabolic fuels in adipocytes

doi: 10.1371/journal.pbio.2006519

Figure Lengend Snippet: (A) Schematic of the Cas9/sgRNA targeting of ATP7A. The sgRNA-targeting sequence is shown; the PAM sequence is labeled in red. The results of sequencing of ATP7A genomic regions are shown below the WT sequence. (B) Protein levels of ATP7A in undifferentiated WT and ATP7A +/− ; α-tubulin is a loading control. (C) The total Cu levels in undifferentiated WT and ATP7A +/− and ATP7A −/− cells measured using atomic absorption spectroscopy ( n = 3). (D) The XFM images of phosphate (“P”), used as a control, and Cu in undifferentiated WT and ATP7A +/− cells show differences in the intracellular distribution of Cu ( n = 3); nuclei are indicated by orange circles. (E) The quantitative analysis of fluorescence intensity in the perinuclear region containing bright Cu puncta in WT and ATP7A +/− cells ( n = 8, 3 respectively). (F) The bright-field images of differentiated WT and ATP7A +/− cells at day 9 illustrate enlargement of cells with down-regulated ATP7A. (G) Distribution of cell sizes and (H) triglyceride levels for WT ( n = 98; n = 3) and ATP7A +/− adipocytes ( n = 101; n = 3); triglyceride levels were normalized to the protein levels, and these values were compared to WT cells’ values taken as 1; the ratio is plotted. Underlying data can be found in ; Student’s t test, **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, ns p > 0.05. The data are presented as mean ± SEM, and median ± IQR for cell distribution. Cas9, CRISPR-associated 9; Cu, copper; ns, not significant; PAM, protospacer-adjacent motif; sgRNA, single guide RNA; WT, wild type; XFM, X-ray fluorescence microscopy.

Article Snippet: Primary antibodies used in immunoblotting were mouse monoclonal anti-rabbit anti-ATP7A CT77 (Hycult biotech), mouse monoclonal anti-tubulin (Sigma, T8203), mouse monoclonal anti-SSAO 7–106 (AK 982/02), mouse anti-Syn6 (BD Transduction Laboratories, 610636), and goat polyclonal anti-mouse IgG Alexa488-conjugate (Molecular Probes, A-11001).

Techniques: Sequencing, Labeling, Atomic Absorption Spectroscopy, Fluorescence, CRISPR, Microscopy

Journal: PLoS Biology

Article Title: Copper-dependent amino oxidase 3 governs selection of metabolic fuels in adipocytes

doi: 10.1371/journal.pbio.2006519

Figure Lengend Snippet: (A) Cu limitation with 10 μM TTM for 48 h decreases the SSAO activity ( n = 3). (B) SSAO activity in the epididymal adipose tissue from 13-wk-old male rats fed with Cu-adequate or low-Cu diet for 8 wk ( n = 7). (C) Down-regulation of ATP7A in ATP7A +/− adipocytes causes a decrease in SSAO activity ( n = 4). (D) Immunocytochemistry shows that SSAO transits the ATP7A-containing compartment on its way to the plasma membrane. Top: ATP7A (green) is localized to the TGN, as evidenced by its colocalization with the TGN marker Syn6 (red); Bottom: SSAO (red) is present at the plasma membrane and inside the cells, where it colocalizes with ATP7A (green). Underlying data can be found in ; Student’s t test, **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, ns p > 0.05. Cu, copper; ns, not significant; SSAO, semicarbazide-sensitive amine oxidase; Syn6, syntaxin 6; TGN, trans -Golgi network; TTM, tetrathiomolybdate; WT, wild type.

Article Snippet: Primary antibodies used in immunoblotting were mouse monoclonal anti-rabbit anti-ATP7A CT77 (Hycult biotech), mouse monoclonal anti-tubulin (Sigma, T8203), mouse monoclonal anti-SSAO 7–106 (AK 982/02), mouse anti-Syn6 (BD Transduction Laboratories, 610636), and goat polyclonal anti-mouse IgG Alexa488-conjugate (Molecular Probes, A-11001).

Techniques: Activity Assay, Immunocytochemistry, Marker

Journal: PLoS Biology

Article Title: Copper-dependent amino oxidase 3 governs selection of metabolic fuels in adipocytes

doi: 10.1371/journal.pbio.2006519

Figure Lengend Snippet: (A) The Cas9/sgRNA-targeting sites in the SSAO gene. The sgRNA-targeting sequence is shown, and the PAM sequence is labeled in red. The results of sequencing of both AOC3 alleles in SSAO −/− cells are shown under the WT sequence. (B) The SSAO activity in WT and SSAO −/− adipocytes ( n = 4). (C) Immunostaining of SSAO (red) and ATP7A (green) in the WT and SSAO −/− adipocytes. (D) Bright-field images of differentiated WT and SSAO −/− cells at day 9. (E) The size distribution and (F) triglyceride levels for the WT ( n = 133; n = 3) and SSAO −/− ( n = 139; n = 3) adipocytes (triglyceride levels were normalized to protein levels, and the values for SSAO −/− cells were compared to the WT control; the ratio is plotted). (G) The size distribution and (I) triglyceride levels for SSAO −/− cells in the basal medium ( n = 178), after treatment with the 0.6 μg/ml sSSAO ( n = 150; n = 3), or 1 μg/ml sSSAO ( n = 184; n = 3); triglyceride levels were normalized to protein levels, and the values for sSSAO-treated cells were compared to values at basal conditions; the ratio is plotted. (H) Effect of sSSAO on the size of SSAO −/− cells at day 9 of differentiation. Underlying data can be found in ; Student’s t test, **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, ns p > 0.05. The data are presented as mean ± SEM, and median ± IQR for cell distribution. Cas9, CRISPR-associated 9; ns, not significant; PAM, protospacer-adjacent motif; sgRNA, single guide RNA; SSAO, semicarbazide-sensitive amine oxidase; sSSAO, recombinant soluble SSAO; WT, wild type.

Article Snippet: Primary antibodies used in immunoblotting were mouse monoclonal anti-rabbit anti-ATP7A CT77 (Hycult biotech), mouse monoclonal anti-tubulin (Sigma, T8203), mouse monoclonal anti-SSAO 7–106 (AK 982/02), mouse anti-Syn6 (BD Transduction Laboratories, 610636), and goat polyclonal anti-mouse IgG Alexa488-conjugate (Molecular Probes, A-11001).

Techniques: Sequencing, Labeling, Activity Assay, Immunostaining, CRISPR, Recombinant

Journal: PLoS Biology

Article Title: Copper-dependent amino oxidase 3 governs selection of metabolic fuels in adipocytes

doi: 10.1371/journal.pbio.2006519

Figure Lengend Snippet: (A) Schematic of the Cas9/sgRNA targeting of ATP7A. The sgRNA-targeting sequence is shown; the PAM sequence is labeled in red. The results of sequencing of ATP7A genomic regions are shown below the WT sequence. (B) Protein levels of ATP7A in undifferentiated WT and ATP7A +/− ; α-tubulin is a loading control. (C) The total Cu levels in undifferentiated WT and ATP7A +/− and ATP7A −/− cells measured using atomic absorption spectroscopy ( n = 3). (D) The XFM images of phosphate (“P”), used as a control, and Cu in undifferentiated WT and ATP7A +/− cells show differences in the intracellular distribution of Cu ( n = 3); nuclei are indicated by orange circles. (E) The quantitative analysis of fluorescence intensity in the perinuclear region containing bright Cu puncta in WT and ATP7A +/− cells ( n = 8, 3 respectively). (F) The bright-field images of differentiated WT and ATP7A +/− cells at day 9 illustrate enlargement of cells with down-regulated ATP7A. (G) Distribution of cell sizes and (H) triglyceride levels for WT ( n = 98; n = 3) and ATP7A +/− adipocytes ( n = 101; n = 3); triglyceride levels were normalized to the protein levels, and these values were compared to WT cells’ values taken as 1; the ratio is plotted. Underlying data can be found in ; Student’s t test, **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, ns p > 0.05. The data are presented as mean ± SEM, and median ± IQR for cell distribution. Cas9, CRISPR-associated 9; Cu, copper; ns, not significant; PAM, protospacer-adjacent motif; sgRNA, single guide RNA; WT, wild type; XFM, X-ray fluorescence microscopy.

Article Snippet: Proteins were then resolved on 8% Laemmli SDS-PAGE and transferred to PVDF membrane at 90 V for 90 min. Primary antibodies used in immunoblotting were rabbit monoclonal anti-ATP7A CT77 (Hycult biotech), mouse monoclonal anti-tubulin (Sigma, T8203), mouse Anti-Na+/K+ ATPase α-1 Antibody (Millipore 06–520), and rabbit Anti-ATP7b antibody [EPR6794] (ab124973).

Techniques: Sequencing, Labeling, Atomic Absorption Spectroscopy, Fluorescence, CRISPR, Microscopy

Journal: PLoS Biology

Article Title: Copper-dependent amino oxidase 3 governs selection of metabolic fuels in adipocytes

doi: 10.1371/journal.pbio.2006519

Figure Lengend Snippet: (A) Cu limitation with 10 μM TTM for 48 h decreases the SSAO activity ( n = 3). (B) SSAO activity in the epididymal adipose tissue from 13-wk-old male rats fed with Cu-adequate or low-Cu diet for 8 wk ( n = 7). (C) Down-regulation of ATP7A in ATP7A +/− adipocytes causes a decrease in SSAO activity ( n = 4). (D) Immunocytochemistry shows that SSAO transits the ATP7A-containing compartment on its way to the plasma membrane. Top: ATP7A (green) is localized to the TGN, as evidenced by its colocalization with the TGN marker Syn6 (red); Bottom: SSAO (red) is present at the plasma membrane and inside the cells, where it colocalizes with ATP7A (green). Underlying data can be found in ; Student’s t test, **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, ns p > 0.05. Cu, copper; ns, not significant; SSAO, semicarbazide-sensitive amine oxidase; Syn6, syntaxin 6; TGN, trans -Golgi network; TTM, tetrathiomolybdate; WT, wild type.

Article Snippet: Proteins were then resolved on 8% Laemmli SDS-PAGE and transferred to PVDF membrane at 90 V for 90 min. Primary antibodies used in immunoblotting were rabbit monoclonal anti-ATP7A CT77 (Hycult biotech), mouse monoclonal anti-tubulin (Sigma, T8203), mouse Anti-Na+/K+ ATPase α-1 Antibody (Millipore 06–520), and rabbit Anti-ATP7b antibody [EPR6794] (ab124973).

Techniques: Activity Assay, Immunocytochemistry, Marker

Journal: PLoS Biology

Article Title: Copper-dependent amino oxidase 3 governs selection of metabolic fuels in adipocytes

doi: 10.1371/journal.pbio.2006519

Figure Lengend Snippet: (A) The Cas9/sgRNA-targeting sites in the SSAO gene. The sgRNA-targeting sequence is shown, and the PAM sequence is labeled in red. The results of sequencing of both AOC3 alleles in SSAO −/− cells are shown under the WT sequence. (B) The SSAO activity in WT and SSAO −/− adipocytes ( n = 4). (C) Immunostaining of SSAO (red) and ATP7A (green) in the WT and SSAO −/− adipocytes. (D) Bright-field images of differentiated WT and SSAO −/− cells at day 9. (E) The size distribution and (F) triglyceride levels for the WT ( n = 133; n = 3) and SSAO −/− ( n = 139; n = 3) adipocytes (triglyceride levels were normalized to protein levels, and the values for SSAO −/− cells were compared to the WT control; the ratio is plotted). (G) The size distribution and (I) triglyceride levels for SSAO −/− cells in the basal medium ( n = 178), after treatment with the 0.6 μg/ml sSSAO ( n = 150; n = 3), or 1 μg/ml sSSAO ( n = 184; n = 3); triglyceride levels were normalized to protein levels, and the values for sSSAO-treated cells were compared to values at basal conditions; the ratio is plotted. (H) Effect of sSSAO on the size of SSAO −/− cells at day 9 of differentiation. Underlying data can be found in ; Student’s t test, **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, ns p > 0.05. The data are presented as mean ± SEM, and median ± IQR for cell distribution. Cas9, CRISPR-associated 9; ns, not significant; PAM, protospacer-adjacent motif; sgRNA, single guide RNA; SSAO, semicarbazide-sensitive amine oxidase; sSSAO, recombinant soluble SSAO; WT, wild type.

Article Snippet: Proteins were then resolved on 8% Laemmli SDS-PAGE and transferred to PVDF membrane at 90 V for 90 min. Primary antibodies used in immunoblotting were rabbit monoclonal anti-ATP7A CT77 (Hycult biotech), mouse monoclonal anti-tubulin (Sigma, T8203), mouse Anti-Na+/K+ ATPase α-1 Antibody (Millipore 06–520), and rabbit Anti-ATP7b antibody [EPR6794] (ab124973).

Techniques: Sequencing, Labeling, Activity Assay, Immunostaining, CRISPR, Recombinant

Journal: The Journal of Biological Chemistry

Article Title: The Activity of Menkes Disease Protein ATP7A Is Essential for Redox Balance in Mitochondria

doi: 10.1074/jbc.M116.727248

Figure Lengend Snippet: Deletion of ATP7A in 3T3-L1 preadipocytes is associated with elevation of cellular copper content and increased oxidation of glutathione in mitochondria. A , CRISPR/Cas9 targeting of exon 2 generated deletion in both alleles of genomic ATP7A in 3T3-L1 cells. B , Western blotting analysis of cell lysates shows the presence of ATP7A in the WT 3T3-L1 cells and a loss of ATP7A expression in CRISPR/Cas9-targeted 3T3-L1 cells. α-Tubulin was used as a loading control. C , copper levels in wild-type (3.4 ± 0.49 ng/mg protein) and ATP7A −/− 3T3-L1 (10.32 ± 0.41 ng/mg protein) cells under basal conditions (****, p < 0.0001; n = 3). D , ratiometric (IR 405/488 ) false-colored image of MTS-GRX1-roGFP2 fluorescence in WT and ATP7A −/− 3T3-L1. Cells were imaged at ×40 magnification. E , oxidation of MTS-GRX1-roGFP2 in mitochondria of wild-type (28.08 ± 17.39, n = 20 cells) and ATP7A −/− 3T3-L1 cells (44.90 ± 20.73, n = 28 cells). **, p = 0.0049. Data are mean ± S.E. of three independent experiments.

Article Snippet: 20 μg of homogenate was separated on 7.5% polyacrylamide gels, transferred onto a PVDF membrane, and incubated overnight at 4 °C with rabbit anti-ATP7A CT77 (Hycult Biotech) to detect ATP7A and with mouse anti-tubulin (Sigma), used as a loading control.

Techniques: CRISPR, Generated, Western Blot, Expressing, Fluorescence