Journal: International Journal of Molecular Sciences

Article Title: ATP Binding Cassette Transporter A1 is Involved in Extracellular Secretion of Acetylated APE1/Ref-1

doi: 10.3390/ijms20133178

Figure Lengend Snippet: ATP binding cassette transporter A1 (ABCA1) was involved in the secretion of APE1/Ref-1. ( A ) The subfamily of ABC transporter expressed in HEK293T cells. The qRT-PCR analysis for ABC transporters using lysates from HEK293T cells. β-actin was tested in parallel and normalized an equal amounts of total RNA per sample. ( B ) The subfamily of ABC transporter expressed in HEK293T cells transiently transfected with siRNA. (Left) The qRT-PCR analysis for knock-down of ABC transporters in transfected cells with each siRNA. (Right) mRNA level of each ABC transporters were analyzed using qRT-PCR in siABCA1 transfected cells. Columns , mean ( n = 3); bars , SE. ** p < 0.01 indicated a significantly different result from non-specific siRNA (NC) transfected cells by two-way ANOVA followed by Bonferroni’s multiple comparison test. ( C ) Changes in the total amount of secreted Ac-APE1/Ref-1 from cells after knock-down of each ABC transporter were analyzed using ELISA. Columns , mean ( n = 3); bars , SE. # p < 0.05 indicated a significantly different result from TSA treated cells or between groups by two-way ANOVA followed by Bonferroni’s multiple comparison test.

Article Snippet: A5316, St. Louis, MO, USA); Monoclonal anti-ABCA1 antibody (cat.no. sc-58219, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and polyclonal antibody (cat.no.

Techniques: Binding Assay, Quantitative RT-PCR, Transfection, Knockdown, Comparison, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: ATP Binding Cassette Transporter A1 is Involved in Extracellular Secretion of Acetylated APE1/Ref-1

doi: 10.3390/ijms20133178

Figure Lengend Snippet: Plasma membrane associated APE1/Ref-1 is bound to ABCA1 in response to acetylation. Cells transiently expressing wild type APE1/Ref-1-FLAG or mutant APE1/Ref-1(K6/7R)-FLAG were treated with 1 µM TSA for 1 h. ( A ) Whole cell lysates were immunoprecipitated using the monoclonal anti-ABCA1 antibody, followed by immunoblot with the anti-FLAG antibody. ( B ) For reverse immunoprecipitation, cell lysates were immunoprecipitated with anti-APE1/Ref-1 antibody followed by immunoblot analysis with the polyclonal anti-ABCA1 antibody. Blots were stripped and re-probed with anti-ABCA1 or FLAG antibodies to ensure equal protein loading and no contamination of cellular proteins. Similar results were observed in replicate experiments. Columns , mean ( n = 2-3); bars , SE. *, p < 0.05 indicates a significantly different result from control cells according to unpaired t- tests. ( C ) The binding between APE1/Ref-1 and ABCA1 in the plasma membrane was visualized using with a Duolink II PLA system with primary polyclonal anti-APE1/Ref-1 and monoclonal anti-ABCA1 antibodies (PLA † ). The PLA-specific fluorescence which represents the APE1/Ref-1-ABCA1 signal, and the DAPI nuclear staining are in red and blue, respectively. The experiment was repeated multiple times with similar results; the data shown here are from a representative experiment. Optical slices were examined using a 40× oil immersion objective with a 2× zoom factor. Scale bar, 20 µm (×80).

Article Snippet: A5316, St. Louis, MO, USA); Monoclonal anti-ABCA1 antibody (cat.no. sc-58219, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and polyclonal antibody (cat.no.

Techniques: Clinical Proteomics, Membrane, Expressing, Mutagenesis, Immunoprecipitation, Western Blot, Control, Binding Assay, Proximity Ligation Assay, Fluorescence, Staining

Journal: International Journal of Molecular Sciences

Article Title: ATP Binding Cassette Transporter A1 is Involved in Extracellular Secretion of Acetylated APE1/Ref-1

doi: 10.3390/ijms20133178

Figure Lengend Snippet: The siRNA sequences of ABC transporters.

Article Snippet: A5316, St. Louis, MO, USA); Monoclonal anti-ABCA1 antibody (cat.no. sc-58219, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and polyclonal antibody (cat.no.

Techniques: Sequencing

Journal: International Journal of Molecular Sciences

Article Title: ATP Binding Cassette Transporter A1 is Involved in Extracellular Secretion of Acetylated APE1/Ref-1

doi: 10.3390/ijms20133178

Figure Lengend Snippet: The primers for glyburide-sensitive human ABC transporters.

Article Snippet: A5316, St. Louis, MO, USA); Monoclonal anti-ABCA1 antibody (cat.no. sc-58219, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and polyclonal antibody (cat.no.

Techniques:

Journal: Molecular Brain

Article Title: Axl receptor tyrosine kinase is a regulator of apolipoprotein E

doi: 10.1186/s13041-020-00609-1

Figure Lengend Snippet: AZ7235 increases ABCA1 expression. ( a ) Cellular ABCA1 protein levels were measured by immunoblot in CCF-STTG1 cells after 72 h treatment with DMSO, 1 μM T0901317, or 3 μM AZ7235. Representative blot was generated from same gel shown in Fig. f. ( b ) CCF-STTG1 cells were labeled with 3 H-cholesterol with co-treatment of DMSO alone, 1 μM T0901317, or 3 μM AZ7235 for 24 h. Cholesterol efflux over 24 h in the absence (NA) or presence of 10 μg/ml of lipid-free apoA-I along with the above drug treatment was evaluated. Graphs represent mean % efflux and standard deviation of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 comparing drug effect over respective DMSO control; ### P < 0.001 comparing between NA vs. apoA-I within each drug condition by blocked three-way ANOVA post-hoc tests. ( c ) Particle size distribution of apoE-containing lipoproteins in the unconcentrated 72-h-conditioned media from drug-treated CCF-STTG1 were assessed by 6% native PAGE followed by immunoblotting for apoE. The ladder on the left represents Stokes diameter. ( d - f ) Cellular ABCA1 protein levels were measured by immunoblot in primary human astrocytes, HMC3 and primary human brain vascular pericytes after 72 h treatment with vehicle control DMSO, 1 μM T0901317, 3 μM AZ7235. Graphs represent fold-change over DMSO control (dashed line) and +/− 95% confidence intervals from N independent experiments indicated in brackets. *** P < 0.001 compared to vehicle control using blocked two-way ANOVA post-hoc tests. Immunoblot images were cropped to show relevant lanes

Article Snippet: After blocking with 5% non-fat milk in PBS for 1 h, membranes were probed for 1 h at 4 °C with 1:1000 rabbit-anti-apoE (Cell Signaling Technology, cat# 13366), 1:1000 monoclonal rabbit-anti-ABCA1 [Clone 1276B] (Novus, cat# NBP2–54792), 1:1000 rabbit-anti-Axl (Cell Signaling Technology, cat# 8661).

Techniques: Expressing, Western Blot, Generated, Labeling, Standard Deviation, Clear Native PAGE

Journal: Molecular Brain

Article Title: Axl receptor tyrosine kinase is a regulator of apolipoprotein E

doi: 10.1186/s13041-020-00609-1

Figure Lengend Snippet: AZ7235-mediated apoE and ABCA1 induction is LXR-independent. U2-OS LXR-Gal4 chimeric Luciferase reporter assays were used to demonstrate that AZ7235 (1.5 nM – 30 μM) have no direct LXRα ( a ) or LXRβ ( b ) agonist activity, unlike the direct LXR agonist T091317, after 40 h of treatment. ( c ) Vehicle control, DMSO, 1 μM T0901317, 3 μM AZ7235 were added to CCF-STTG1 cells transfected with a mixture of LXR-responsive Firefly luciferase construct and constitutively expressing Renilla luciferase construct as an internal control. Luciferase activities were measured after 24 h of treatment. Error bars represent standard deviation from technical triplicates. *** P < 0.0001 by ANOVA post-hoc analysis. ( d - e ) LXR-knockout (LXRα−/β-) and LXRα-expressing (LXRα+/β-) MEF cells were treated with DMSO or drugs for 48 h. Apoe and Abca1 mRNA levels were measured by qRT-PCR. Graph represents fold-change over respective DMSO control (dash line) +/− 95% CI from 5 experiments

Article Snippet: After blocking with 5% non-fat milk in PBS for 1 h, membranes were probed for 1 h at 4 °C with 1:1000 rabbit-anti-apoE (Cell Signaling Technology, cat# 13366), 1:1000 monoclonal rabbit-anti-ABCA1 [Clone 1276B] (Novus, cat# NBP2–54792), 1:1000 rabbit-anti-Axl (Cell Signaling Technology, cat# 8661).

Techniques: Luciferase, Activity Assay, Transfection, Construct, Expressing, Standard Deviation, Knock-Out, Quantitative RT-PCR

Journal: Molecular Brain

Article Title: Axl receptor tyrosine kinase is a regulator of apolipoprotein E

doi: 10.1186/s13041-020-00609-1

Figure Lengend Snippet: AZ7235 has nominal effects on secretion and mRNA levels of apoE and no effect on ABCA1 expression in primary murine mixed glia expressing human apoE3. Primary mixed glial cells (~ 90% astrocytes, 10% microglia) were cultivated from neonatal human APOE3 targeted-replacement mice. Cells were re-plated at 18–20 days in vitro followed by treatment with vehicle (DMSO), 1 μM T0901317, 1 μM and 3 μM AZ7235 for 72 h. ( a ) Secreted apoE levels in conditioned media were measured by apoE ELISA. The graph represents mean concentration and standard deviation from 4 independent experiments. ( b ) Representative immunoblot of cellular ABCA1 and apoE with GAPDH as the loading control. Immunoblot images were cropped to show relevant lanes. ( c ) Quantification of cellular apoE and ( d ) ABCA1 protein levels in murine glia lysates by immunoblotting. ( e - f ) Human APOE ( e ) and mouse Abca1 ( f ) mRNA levels were measured by qRT-PCR. Graphs represent fold-change over DMSO control (dashed line) and +/− 95% confidence intervals from N independent experiments indicated in brackets. ** P < 0.01, *** P < 0.001 compared to vehicle control by blocked ANOVA post-hoc tests

Article Snippet: After blocking with 5% non-fat milk in PBS for 1 h, membranes were probed for 1 h at 4 °C with 1:1000 rabbit-anti-apoE (Cell Signaling Technology, cat# 13366), 1:1000 monoclonal rabbit-anti-ABCA1 [Clone 1276B] (Novus, cat# NBP2–54792), 1:1000 rabbit-anti-Axl (Cell Signaling Technology, cat# 8661).

Techniques: Expressing, In Vitro, Enzyme-linked Immunosorbent Assay, Concentration Assay, Standard Deviation, Western Blot, Quantitative RT-PCR

Journal: Frontiers in Immunology

Article Title: Metabolic-related gene pairs signature analysis identifies ABCA1 expression levels on tumor-associated macrophages as a prognostic biomarker in primary IDH WT glioblastoma

doi: 10.3389/fimmu.2022.869061

Figure Lengend Snippet: The Expression Level of ABCA1 in TAMs Positively Correlated with the Population of TAMs with IDH WT GBM Tumor Progression. (A) Experimental setup to study the links between MRGs expression in macrophage and tumor progression. (B) Quantitative analysis of mRNA expression levels of 5 MRGs in TAMs with tumor progression. C57 mice were intracranially inoculated with GL261 cells. On day 7, day 14 and day 21, mice were sacrificed and collected tumor tissues. TAMs were isolated from tumors by anti-F4/80 microbeads and subjected to qPCR to detect mRNA expression (n = 3). (C) Immunohistochemical analysis of the expression levels of ABCA1 in mouse tumor and normal brain tissue. (D, E) Representative flow cytometry plots (D) and quantitative analysis (E) of TAMs and peripherally splenic monocytes/macrophage ABCA1 expression levels with tumor progression. Orthotopic tumors were collected on days 7, 14 and 21 after tumor inoculation. n = 8. f-g: Quantitative analysis of TAM populations (F) and pearson correlation analysis of TAM populations and TAM ABCA1 expression levels (G) . Data is related to (D, E) . The difference between risk groups was calculated by the Mann–Whitney test (NS, no statistical significance, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: Dissociated cells were further incubated with the following antibodies: anti-mouse CD16/CD32 (Multi Sciences, clone 2.4G2, Cat No. AM016-100), IgG2a, κ isotype ctrl (Biolegend, clone MOPC-173, Cat No. 400233), anti-mouse ABCA1 (BIO-RAD, clone 5A1-1422, Cat No. MCA2681), anti-mouse F4/80 (Biolegend, clone BM8, Cat No. 123110), anti-mouse\human CD11b CM1/70, Cat No. 101229), anti-mouse CD86 (Biolegend, clone GL-1, Cat No. 105005), anti-mouse CD206 (Biolegend, clone C068C2, Cat No. 141715), anti-mouse TNF-α (Biolegend, clone MP6-XT22, Cat No. 506303), anti-mouse IFN-γ (Biolegend, clone XMG1.2, Cat No. 506303), anti-mouse Arginase 1 (Abbexa, Polyclonal, Cat No. abx319179), and anti-mouse CD45 (Biolegend, clone 30-F11, Cat No. 103112).

Techniques: Expressing, Isolation, Immunohistochemical staining, Flow Cytometry, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Metabolic-related gene pairs signature analysis identifies ABCA1 expression levels on tumor-associated macrophages as a prognostic biomarker in primary IDH WT glioblastoma

doi: 10.3389/fimmu.2022.869061

Figure Lengend Snippet: Pharmacological Inhibition of ABCA1 Enhances the Inflammatory Polarization of TAMs In Vivo (A) : Quantification of ABCA1 expression in cholesterol-treated BMDMs ex vivo. BMDMs were treated with the indicated concentrations of cholesterol for 24 h. Cells were harvested and FCM was performed to identify the expression level of ABCA1 (n = 3). (B, C) : Quantification of ABCA1 expression (B) and CD86 and CD206 expression (C) in lovastatin-treated TAMs ex vivo. TAMs were treated with the indicated concentrations of lovastatin for 24 h. FCM was performed to identify these molecular expressions. (D) : Experimental setup of lovastatin-treated murine IDH WT GBM model. Eight mice per group. (E, F) : Representative flow cytometry plots (E) and quantification of ABCA1 expression levels in TAMs (F) . (G–J) : Representative flow cytometry plots (G) and quantification of TAM functional polarization (H–J) . ARG1 (H) were identified as anti-inflammatory macrophage markers; IFN-γ (I) and TNF-α (J) were identified as inflammatory macrophage markers. (K–M) : Experimental setup (K) and tumor growth and survival monitoring (L, M) of lovastatin-treated murine GL261 IDH-WT-Luc model. Eight mice per group. The difference between risk groups was calculated by the Mann–Whitney test and log-rank test (NS, no statistical significance, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: Dissociated cells were further incubated with the following antibodies: anti-mouse CD16/CD32 (Multi Sciences, clone 2.4G2, Cat No. AM016-100), IgG2a, κ isotype ctrl (Biolegend, clone MOPC-173, Cat No. 400233), anti-mouse ABCA1 (BIO-RAD, clone 5A1-1422, Cat No. MCA2681), anti-mouse F4/80 (Biolegend, clone BM8, Cat No. 123110), anti-mouse\human CD11b CM1/70, Cat No. 101229), anti-mouse CD86 (Biolegend, clone GL-1, Cat No. 105005), anti-mouse CD206 (Biolegend, clone C068C2, Cat No. 141715), anti-mouse TNF-α (Biolegend, clone MP6-XT22, Cat No. 506303), anti-mouse IFN-γ (Biolegend, clone XMG1.2, Cat No. 506303), anti-mouse Arginase 1 (Abbexa, Polyclonal, Cat No. abx319179), and anti-mouse CD45 (Biolegend, clone 30-F11, Cat No. 103112).

Techniques: Inhibition, In Vivo, Expressing, Ex Vivo, Flow Cytometry, Functional Assay, MANN-WHITNEY