Journal: BioMed Research International

Article Title: Liver X Receptor Agonist TO901317 Attenuates Paraquat-Induced Acute Lung Injury through Inhibition of NF- κ B and JNK/p38 MAPK Signal Pathways

doi: 10.1155/2017/4652695

Figure Lengend Snippet: The expression of ABCA1 in lung tissues. We collected the lung tissues to perform Western blot to evaluate the expression of ABCA1 at 72 h after PQ administration. TO901317L: TO901317 at the low dose of 5 mg/kg; TO901317H: TO901317 at the high dose of 20 mg/kg. The values presented are the mean ± SD ( n = 6). ∗ p < 0.05 versus control group, △ p < 0.05 versus TO901317L group, # p < 0.05 versus PQ group, and ☆ p < 0.05 versus PQ + TO901317L group.

Article Snippet: Subsequently, proteins were transferred onto polyvinylidene difluoride membranes and incubated at 4°C overnight with primary antibodies (all from Abcam, Cambridge, UK, except as noted) specific for ABCA1 (1 : 500), NF- κ Bp65 (1 : 400), I κ B- α (1 : 1000), phosphor-p38 (1 : 1000 CST, Danvers, MA, USA), phosphor-JNK (1 : 1000), Bax (1 : 2000), and Bcl-2 (1 : 400).

Techniques: Expressing, Western Blot

Journal: BMC Neuroscience

Article Title: Cholesterol efflux regulator ABCA1 exerts protective role against high shear stress-induced injury of HBMECs via regulating PI3K/Akt/eNOS signaling

doi: 10.1186/s12868-022-00748-2

Figure Lengend Snippet: HSS alters cellular functions and modulates the ABCA1 expression and PI3K/Akt/eNOS signaling in HBMEC. A Effect of SS on the viability of HBMECs. B Effect of SS on the caspase 3/7 activity in HBMECs. C Images of flow cytometry analysis of the apoptosis of HBMECs and related numerical quantification. D Images of Transwell analysis of the migration of HBMECs and related numerical quantification. E NO production in HBMECs. F ROS production in HBMECs. G ELISA detection of the release of inflammatory cytokines from HBMECs. H qRT-PCR detection of the inflammatory cytokines from HBMECs. I qRT-PCR detection of ABCA1, MMP9, AQP4, and CYP46 from HBMECs. J qRT-PCR detection of PI3K, AKT and eNOS from HBMECs. K Western blot images showing the expression of ABCA1, MMP9, AQP4, and CYP46 proteins in different groups. L Densitometry analysis of western blot images of ABCA1, MMP9, AQP4, and CYP46. M Western blot images showing the expression of PI3K, AKT and eNOS and their phosphorylated forms in different groups. N Densitometry analysis of western blot images of PI3K, AKT and eNOS and their phosphorylated forms. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 versus control or between indicated compared group; # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.001 versus PSS group. Full-length blots/gels are presented in Additional file : Figures S1 and S2

Article Snippet: Antibodies against phospho-PI3 Kinase p85 (Tyr458)/p55(Tyr199) (CST4228), phospho-Akt (Ser473) (CST4060) and phospho-eNOS (Ser1177) (CST9570) were purchased from Cell Signaling Technology (Waltham, USA). siRNA targeting ABCA1, and corresponding negative controls were purchased from Sangon Biotech (Shanghai, China).

Techniques: Expressing, Activity Assay, Flow Cytometry, Migration, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Western Blot

Journal: BMC Neuroscience

Article Title: Cholesterol efflux regulator ABCA1 exerts protective role against high shear stress-induced injury of HBMECs via regulating PI3K/Akt/eNOS signaling

doi: 10.1186/s12868-022-00748-2

Figure Lengend Snippet: ABCA1 overexpression counteracts HSS-induced functional alterations in HBMECs. A qRT-PCR detection of the effect of ABCA1 overexpression vector on ABCA1 mRNA expression. B qRT-PCR detection of the effect of ABCA1-siRNA on ABCA1 mRNA expression. C Western blot detection of the effect of ABCA1-siRNA and expression vector on ABCA1 mRNA expression. D Densitometry analysis of western blot detection of the effect of ABCA1-siRNA and expression vector on ABCA1 protein expression. E Effect of ABCA1 on the viability of HBMECs under HSS treatment. F Effect of ABCA1 on the caspase 3/7 activity in HBMECs under HSS treatment. G Effect of ABCA1 on the apoptosis of HBMECs under HSS treatment as determined by flow cytometry. H Effect of ABCA1 on ROS production in HBMECs under HSS treatment. I Effect of ABCA1 on NO production in HBMECs under HSS treatment. J Effect of ABCA1 on the release of inflammatory cytokines in HBMECs under HSS treatment. K Effect of ABCA1 on the mRNA expression levels of inflammatory cytokines in HBMECs under HSS treatment as indicated by qRT-PCR experiment. L Effect of ABCA1 on the migration of HBMECs under HSS treatment. ** p < 0.01 and *** p < 0.001 among the compared groups. Full-length blots/gels are presented in Additional file : Figure S3

Article Snippet: Antibodies against phospho-PI3 Kinase p85 (Tyr458)/p55(Tyr199) (CST4228), phospho-Akt (Ser473) (CST4060) and phospho-eNOS (Ser1177) (CST9570) were purchased from Cell Signaling Technology (Waltham, USA). siRNA targeting ABCA1, and corresponding negative controls were purchased from Sangon Biotech (Shanghai, China).

Techniques: Over Expression, Functional Assay, Quantitative RT-PCR, Plasmid Preparation, Expressing, Western Blot, Activity Assay, Flow Cytometry, Migration

Journal: BMC Neuroscience

Article Title: Cholesterol efflux regulator ABCA1 exerts protective role against high shear stress-induced injury of HBMECs via regulating PI3K/Akt/eNOS signaling

doi: 10.1186/s12868-022-00748-2

Figure Lengend Snippet: ABCA1 overexpression counteracts HSS-induced gene regulation changes in HBMECs. A Effect of ABCA1 overexpression and siRNA on the mRNA levels of MMP9, AQP4 and CYP46 as determined by qRT-PCR. B Effect of ABCA1 overexpression and siRNA on the mRNA levels of PI3K, AKT and eNOS as determined by qRT-PCR. C Effect of ABCA1 overexpression and siRNA on the protein levels of MMP9, AQP4, CYP46 and their phosphorylated forms as determined by Western blotting. D Densitometry analysis of MMP9, AQP4 and CYP46. E Effect of ABCA1 overexpression and siRNA on the protein levels of PI3K, AKT and eNOS. F Densitometry analysis of PI3K, AKT and eNOS and their phosphorylated forms. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 among the compared groups. Full-length blots/gels are presented in Additional file : Figures S4 and S5

Article Snippet: Antibodies against phospho-PI3 Kinase p85 (Tyr458)/p55(Tyr199) (CST4228), phospho-Akt (Ser473) (CST4060) and phospho-eNOS (Ser1177) (CST9570) were purchased from Cell Signaling Technology (Waltham, USA). siRNA targeting ABCA1, and corresponding negative controls were purchased from Sangon Biotech (Shanghai, China).

Techniques: Over Expression, Quantitative RT-PCR, Western Blot

Journal: BMC Neuroscience

Article Title: Cholesterol efflux regulator ABCA1 exerts protective role against high shear stress-induced injury of HBMECs via regulating PI3K/Akt/eNOS signaling

doi: 10.1186/s12868-022-00748-2

Figure Lengend Snippet: Primer sequences

Article Snippet: Antibodies against phospho-PI3 Kinase p85 (Tyr458)/p55(Tyr199) (CST4228), phospho-Akt (Ser473) (CST4060) and phospho-eNOS (Ser1177) (CST9570) were purchased from Cell Signaling Technology (Waltham, USA). siRNA targeting ABCA1, and corresponding negative controls were purchased from Sangon Biotech (Shanghai, China).

Techniques: Sequencing

Journal: bioRxiv

Article Title: AUF-1 knock down in mice overarches butyrate driven hypo-cholesteraemia by conjuring AUF-1-Dicer-1-miR122 hierarchy

doi: 10.1101/2022.06.13.496022

Figure Lengend Snippet: Hepatic expression of cholesterol metabolic pathway: hmgcr , hmgcs , dhcr7 , acat-2 and cyp7A1 was studied in all the groups by qPCR (A). TEM images of the liver sections showing lipid droplets. The sections were visualized under a FEI Tecnai 12 Biotwin transmission electron microscope (FEI, Hillsboro, OR, USA) at an accelerating voltage of 100 kV. Scale bar in the image is 2µm (B). Analysis of hepatic expression of HMGCR, ABCA1 and ABCA5 by western blot (C). Densitometry was done using ImageJ showing relative expression with respect to β-actin control (D). N = 5 / group, data is represented as mean ±SE. The experiment was repeated thrice. *** represents p<0.001, ** represents p<0.01, * represents p<0.05, ns represents not significant.

Article Snippet: Prime script first strand cDNA synthesis kit, TB Green Premix ex-Taq (Tli RNase H+) qPCR kit were purchased from Takara (Shiga, Japan). (Ripa lysis buffer, anti-β-actin antibody (polyclonal), siAUF1, Anti-AUF1 antibody (rabbit polyclonal), anti-HMGCR antibody (polyclonal), anti-ABCA1 antibody (rabbit monoclonal) were purchased from Cell Signalling Technology (Danvers, MA, USA).

Techniques: Expressing, Transmission Assay, Microscopy, Western Blot

Journal: bioRxiv

Article Title: AUF-1 knock down in mice overarches butyrate driven hypo-cholesteraemia by conjuring AUF-1-Dicer-1-miR122 hierarchy

doi: 10.1101/2022.06.13.496022

Figure Lengend Snippet: Analysis of expression of ABCA1 and ABCA5 in Huh7 cells by western blot as a function of butyrate concentration (A). The corresponding densitometry using ImageJ showing relative expression of ABCA1 and ABCA5 with respect to β-actin control (B). Percent cholesterol efflux as function of butyrate concentration in the form of 22-NBD-cholesterol was monitored by measuring 22-NBD fluorescence. Huh7 cells were loaded with liposomal 22-NBD-cholesterol for 24 h and subsequently treated with 20mM butyrate. The cells were washed and equilibrated in serum free medium for 18h. Thereafter the cells were treated with or without HDL (1 µg/ml, 5 µg/ml and 20 µg/ml). The fluorescence intensity (FI) of 22-NBD-cholesterol in the medium and cell lysate was detected by MT-600F fluorescence microplate reader (Corona Electric, Hitachinaka, Japan) using 469 nm excitation and 537 nm emission filters in a black polystyrene 96-well plate. The efflux (%) was calculated as (FI sup X 100)/ (FI sup + FI cell lysate ) (C). Expression of pre-miR122, miR122, Dicer1 and AUF1 (D) after butyrate or propionate or aceatate treatment 20 mM each as measured by qPCR. N ≥3 for all data sets, data is represented as mean ±SE. *** represents p<0.001, ** represents p<0.01, * represents p<0.05, ns represents not significant.

Article Snippet: Prime script first strand cDNA synthesis kit, TB Green Premix ex-Taq (Tli RNase H+) qPCR kit were purchased from Takara (Shiga, Japan). (Ripa lysis buffer, anti-β-actin antibody (polyclonal), siAUF1, Anti-AUF1 antibody (rabbit polyclonal), anti-HMGCR antibody (polyclonal), anti-ABCA1 antibody (rabbit monoclonal) were purchased from Cell Signalling Technology (Danvers, MA, USA).

Techniques: Expressing, Western Blot, Concentration Assay, Fluorescence

Journal: JCI Insight

Article Title: Aldehyde dehydrogenase 2 and PARP1 interaction modulates hepatic HDL biogenesis by LXR α -mediated ABCA1 expression

doi: 10.1172/jci.insight.155869

Figure Lengend Snippet: ( A ) Hepatic mRNA levels of ABCA1 and ALDH2 in WT and AKO mouse tissues at 32 nd week (Western diet for 26 weeks). WT, n = 5; AKO, n = 5. ( Β ) Western blotting analysis of ABCA1 and LXRα expressions in WT and AKO liver tissue. WT, n = 3; AKO, n = 3. ( C ) Hepatic mRNA levels of LXRα and ABCA1 in LKO and ALKO mice at 32nd week (Western diet for 26 weeks). LKO, n = 5; ALKO, n = 5. ( D ) Western blotting analysis of ABCA1 and LXRα expression in LKO and ALKO liver tissues. LKO, n = 3; ALKO, n = 3. ( E ) Western blotting analysis of ABCA1; LXRα expression in LKO and ALKO hepatocytes treated with ox-LDL (50 μg/mL, 16 h). LKO, n = 3; ALKO, n = 3. ( F ) Western blotting analysis of ABCA1 expression in overexpressed ALDH2 (over-AL) hepatocytes treated with ox-LDL (50 μg/mL, 16 h). LKO, n = 3; ALKO, n = 3. ( G ) IHC analysis of ABCA1 expressions in mouse liver sections (LKO, n = 5; ALKO, n = 5; scale bar: 100 μm). Statistical comparisons were made using a 2-tailed Student’s t test. All data are mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Antibodies against poly/mono-ADP ribose (83732S), PARP1 (46D11), and ABCA1 (E7X5G) were purchased from Cell Signaling Technology.

Techniques: Western Blot, Expressing

Journal: JCI Insight

Article Title: Aldehyde dehydrogenase 2 and PARP1 interaction modulates hepatic HDL biogenesis by LXR α -mediated ABCA1 expression

doi: 10.1172/jci.insight.155869

Figure Lengend Snippet: ( A ) Free cholesterol levels in LKO and ALKO mouse hepatocytes treated with PARP1 inhibitor PJ34 (50 μM, 24 h; P-LKO or P-ALKO) or LXRα agonist T0901317 (10 μM, 24 h, T-LKO or T-ALKO) after treating with ox-LDL (50 μg/mL, 16 h).LKO, n = 3; ALKO, n = 3; P-ALKO, n = 3; T-ALKO, n = 3. ( B ) Free cholesterol levels in WT and AKO mouse hepatocytes treated with PJ34 (50 μM, 24 h; P-AKO or P-WT) after ox-LDL treatment (50 μg/mL, 16 h). WT, n = 3; AKO, n = 3; P-AKO, n = 3; T-AKO, n = 3. ( C ) AKO or WT mice were injected with 1× PBS or PJ34 and fed with a Western diet for 8 weeks. ( D ) Representative H&E and Oil Red O staining for mouse liver tissues (WT: WT mice injected with 1× PBS; AKO: ALDH2-KO mice injected with 1× PBS; P-AKO: AKO mice injected with PJ34). WT, n = 10; AKO, n = 10; P-AKO, n = 10; scale bar: 100 μm. ( E ) Free cholesterol in liver tissue and HDL-C in plasma. WT, n = 10; AKO, n = 10; P-AKO, n = 10. ( F ) Western blotting analysis of ABCA1 and PARP1 expression in WD mouse liver tissue. WT, n = 3; AKO, n = 3; P-AKO, n = 3. ( G ) Poly(ADP-ribosyl)ation of LXRα in WT, AKO, and P-AKO mouse liver tissue. Experiments were repeated 3 times. Statistical comparisons were made using 1-way ANOVA followed by Student-Newman-Keuls for multiple-comparison tests. All data are mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Antibodies against poly/mono-ADP ribose (83732S), PARP1 (46D11), and ABCA1 (E7X5G) were purchased from Cell Signaling Technology.

Techniques: Injection, Western Blot, Staining, Expressing

Journal: JCI Insight

Article Title: Aldehyde dehydrogenase 2 and PARP1 interaction modulates hepatic HDL biogenesis by LXR α -mediated ABCA1 expression

doi: 10.1172/jci.insight.155869

Figure Lengend Snippet: ( A ) Western blotting analysis of ABCA1 and PARP1 expression in HL-7702 cells after transfection with siNC or siALDH2 treated with ox-LDL (50 μg/mL, 16 h) ( n = 3). ( B ) ALDH2 knockdown increased nuclear translocation of PARP1 and decreased cytosolic PARP1 ( n = 3). Experiments were repeated 3 times. ( C ) Immunofluorescent analysis of the nuclear fraction of PARP1 in ALDH2 knockdown (si-AL, transfection with siALDH2) or ALDH2 overexpression (over-AL, transfection with Flag-ALDH2 plasmid). Red, ALDH2; green, PARP1; blue DAPI; n = 5; scale bar: 5 μm. ( D ) Diagram of major domains in PARP1: DBD, DNA binding domain; NLS, nuclear localization sequence; BRCT, BRCA1 C-terminus domain; WGR, arginine-rich domain; CD, catalytic domain. The schematics of the His-PARP1 expression plasmid as well as domains with truncated mutants. ( A ) 1–214 aa DBD, ( B ) 215–372 aa NLS, ( C ) 373–476 aa BRCT domain, ( D ) 525–656 aa WGR domain, ( E ) 657–1014 aa CD. ( E ) NLS, BRCT, and CD mediated the association of PARP1 with ALDH2. Pulled down ALDH2 was detected by immunoblotting. ( F ) Deletion of NLS attenuated the interactions of PARP1 with ALDH2. Experiments were repeated 3 times. Statistical comparisons were made using a 2-tailed Student’s t test or 1-way ANOVA followed by Student-Newman-Keuls for multiple-comparison tests. All data are mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Antibodies against poly/mono-ADP ribose (83732S), PARP1 (46D11), and ABCA1 (E7X5G) were purchased from Cell Signaling Technology.

Techniques: Western Blot, Expressing, Transfection, Translocation Assay, Over Expression, Plasmid Preparation, Binding Assay, Sequencing

Journal: JCI Insight

Article Title: Aldehyde dehydrogenase 2 and PARP1 interaction modulates hepatic HDL biogenesis by LXR α -mediated ABCA1 expression

doi: 10.1172/jci.insight.155869

Figure Lengend Snippet: ( A ) Representative H&E and Oil Red O staining for mouse liver fed with a Western diet (WD) for 8 weeks (WT and ALDH2 rs671-KI mice, referred to as rs671). Scale bar: 100 μm. ( B ) HDL-C in plasma at 16 th week (WD for 8 weeks). WT, n = 9; rs671, n = 10. ( C ) Western blotting analysis of ABCA1, ALDH2, LXRα, and SR-B1 expression in mouse liver tissue. WT, n = 3; rs671, n = 3. ( D ) IP results of WT ALDH2 or ALDH2 rs671, PARP1. ( E ) IP results of ALDH2 and PARP1 in human liver tissues. WT, n = 3; rs671, n = 3. Experiments were repeated 3 times. ( F ) ALDH2 rs671 significantly increased nuclear translocation of PARP1 in human liver tissue. WT, n = 3; rs671, n = 3. Statistical comparisons were made using a 2-tailed Student’s t test. All data are mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Antibodies against poly/mono-ADP ribose (83732S), PARP1 (46D11), and ABCA1 (E7X5G) were purchased from Cell Signaling Technology.

Techniques: Staining, Western Blot, Expressing, Translocation Assay

Journal: JCI Insight

Article Title: Aldehyde dehydrogenase 2 and PARP1 interaction modulates hepatic HDL biogenesis by LXR α -mediated ABCA1 expression

doi: 10.1172/jci.insight.155869

Figure Lengend Snippet: ALDH2 regulates hepatic HDL biogenesis and increases expression of ABCA1 through decreasing poly(ADP-ribosyl)ation of LXRα by blocking the NLS of PARP1. In ALDH2-KO or ALDH2*2 liver, attenuated ALDH2/PARP1 interaction increases nuclear translocation of PARP1 and poly(ADP-ribosyl)ation of LXRα, which leads to downregulation of ABCA1 and HDL biogenesis. This mechanism operates in the context of feeding with a Western diet that activates LXRα and PARP1.

Article Snippet: Antibodies against poly/mono-ADP ribose (83732S), PARP1 (46D11), and ABCA1 (E7X5G) were purchased from Cell Signaling Technology.

Techniques: Expressing, Blocking Assay, Translocation Assay, Western Blot

Journal: Communications Biology

Article Title: Macrophage Jak2 deficiency accelerates atherosclerosis through defects in cholesterol efflux

doi: 10.1038/s42003-022-03078-5

Figure Lengend Snippet: a Radioactive cholesterol uptake in BMDM of M-Jak2 KO and WT mice ( n = 4–6/genotype). b Quantification of the number of lipid droplets sized 0.3 µm per field stained with BODIPY, in BMDM untreated or treated with LDL, oxLDL, or combined oxLDL and ApoA1 for 24 h ( n = 4–8/genotype). c – j Representative images of BMDM in the conditions described in ( b ). Scale bar, 6.6 µm. k , l Cholesterol efflux measurements using radioactive cholesterol from BMDM; total unstimulated, in response to ApoB-depleted plasma ( n = 9/group) and to ApoA1 ( n = 8/group). m , n BMDM stimulated with cAMP to induce ABCA1, and total efflux from M-Jak2 KO and WT BMDM in response to ApoB-depleted plasma ( n = 9/group) and to ApoA1 ( n = 8/group). o – r Cholesterol efflux measurements after JAK2 inhibition with AG490 from M-Jak2 KO and WT BMDM ( n = 8/group). Statistical analysis: a , b Multiple t tests with Holm–Sidak correction, k – n two-tailed unpaired t tests, and ( o – r ) two-way ANOVA with Tukey’s multiple comparisons were performed. Data are presented as the mean ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. See also Supplementary Figs. .

Article Snippet: Membranes were probed overnight at 4 °C with a primary antibody: total JAK2 (CST#3230; 1:1000), total ABCA1 (NB400-105; 1:500), total SRB1 (NB400-104; 1:1000), total ABCG1 (NB400-132; 1:500), and β-actin (CST#4967; 1:1000).

Techniques: Staining, Inhibition, Two Tailed Test

Journal: Communications Biology

Article Title: Macrophage Jak2 deficiency accelerates atherosclerosis through defects in cholesterol efflux

doi: 10.1038/s42003-022-03078-5

Figure Lengend Snippet: a – c mRNA level of ABCA1, ABCG1 , and SCARB1 in BMDM under basal conditions, after loading with oxLDL or after loading with oxLDL and treating with TO901317 with ApoA1 to induce efflux ( n = 10–19/group). d , e Cholesterol efflux from BMDM of M-Jak2 KO and WT mice treated with the LXR agonist, TO901317 in response to ApoB-depleted plasma ( n = 8/group) and to purified ApoA1 ( n = 8/group). f – i Representative en face images of Oil-red-O-stained lesser curvature of the aortic arch from M-Jak2 KO and WT mice treated with TO901317 or vehicle while on HCD for 3 weeks starting at 6 weeks of age. Scale bar, 1 mm ( j ) and their quantification ( n = 4–9/group). Statistical analysis: a – e two-way ANOVA with Tukey’s multiple comparisons and ( j ) multiple t tests with Holm–Sidak correction were performed. Data are presented as the mean ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. See also Supplementary Fig. .

Article Snippet: Membranes were probed overnight at 4 °C with a primary antibody: total JAK2 (CST#3230; 1:1000), total ABCA1 (NB400-105; 1:500), total SRB1 (NB400-104; 1:1000), total ABCG1 (NB400-132; 1:500), and β-actin (CST#4967; 1:1000).

Techniques: Purification, Staining