Journal: Science signaling

Article Title: Designer high-density lipoprotein particles enhance endothelial barrier function and suppress inflammation

doi: 10.1126/scisignal.adg9256

Figure Lengend Snippet: (A) Side view of A1M-S1P complex with and without the phospholipids, highlighting the S1P binding pocket in both the ApoM-S1P and ApoA1-fused systems. Monomer 1 - ApoA1 in sienna, linker in brown, and ApoM in beige. Monomer 2 - ApoA1 in sage, linker in turquoise, and ApoM in teal. Detail of the binding pocket in A1M-S1P and ApoM-S1P are shown in the right panel. (B) Projection of protein atoms along the top two dominant eigenvectors (eigenvector 1 - PC1 and eigenvector 2 – PC2) is responsible for over 40% of the dominant collective variances in the structure set, generating 5 clusters for which the three-dimensional structure of the most representative member of each cluster is shown. Permanence times: cluster 1 – 9.6%, cluster 2 – 52.9%, cluster 3 – 18.7%, cluster 4 – 12.2%, cluster 5 – 6.5%. (C) Distribution of the proportion of variance between the PCs.

Article Snippet: The A1M fusion was constructed using plasmids for murine ApoA1 (MR203500) and murine ApoM (MR201811) obtained from OriGene.

Techniques: Binding Assay

Journal: Science signaling

Article Title: Designer high-density lipoprotein particles enhance endothelial barrier function and suppress inflammation

doi: 10.1126/scisignal.adg9256

Figure Lengend Snippet: (A) HMEC-1 cells expressing an NF-κB-luciferase reporter were assayed for TNFα-induced NF-κB reporter activity in the presence of ApoA1, A1M, A1M-S1P, and ApoM-Fc-S1P (N = 3 biological replicates per group). Data are presented as means + S.D. **P < 0.01, ***P < 0.001, ****P < 0.0001 by ordinary two-way ANOVA with Tukey’s multiple comparisons test. TNFα is the reference group. (B) HUVECs were starved for 1 h, pre-treated for 10 minutes with ApoM-Fc-S1P (100 nM), iloprost (200 nM), both ApoM-Fc-S1P and iloprost, A1M (200 μg/mL), A1M-S1P (200 μg/mL), or A1M-iloprost (200 μg/mL) and induced with TNFα (10 ng/mL) for 5 hours. Lysates were subjected to immunoblot analysis for ICAM-1. Quantification of immunoblots was analyzed from 3 biological replicates per group. Data are presented as means ± S.D. *P < 0.05, **P < 0.01, ****P < 0.0001 by ANOVA with post-hoc Holm-Sidak’s multiple comparisons test. (C) Cholesterol efflux in response to human HDL (hHDL), human ApoA1 protein (hApoA1), and bacterial A1M (bA1M) in PMA-induced THP-1 cells (N ≥ 5 independent experiments). Data are presented as means + S.D. *P < 0.05, ***P < 0.001, ****P < 0.0001 by one-way ANOVA with Dunnett’s multiple comparisons test.

Article Snippet: The A1M fusion was constructed using plasmids for murine ApoA1 (MR203500) and murine ApoM (MR201811) obtained from OriGene.

Techniques: Expressing, Luciferase, Activity Assay, Western Blot

Journal: Heliyon

Article Title: First-in-human autologous implantation of genetically modified adipocytes expressing LCAT for the treatment of familial LCAT deficiency

doi: 10.1016/j.heliyon.2022.e11271

Figure Lengend Snippet: Key resources.

Article Snippet: Apolipoprotein A1 , Athens Research & Technology , Cat#16-16-120101.

Techniques: Control, Virus, Retroviral, Plasmid Preparation, Clinical Proteomics, Recombinant, Lysis, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Biomolecules

Article Title: CRISPR/dCas9 Transcriptional Activation of Endogenous Apolipoprotein AI and Paraoxonase 1 in Enterocytes Alleviates Endothelial Cell Dysfunction

doi: 10.3390/biom11121769

Figure Lengend Snippet: Graphical representation of the experimental design. Caco-2 cells were transfected with ApoAI or PON1 CRISPR activation plasmids. Then, 48 h after transfection, the medium was replaced and the cells were incubated for another 24 h with DMEM to obtain the conditioned media enriched in ApoAI or PONI. After this time interval, Caco-2 cells were lysed and further processed for Real-Time PCR and Western blot analysis. The conditioned media from transfected Caco-2 cells were used to evaluate their effects on EC function.

Article Snippet: ApoAI CRISPR activation plasmid (sc-400499-ACT), PON1 CRISPR activation plasmid (sc-402701-ACT), control CRISPR activation plasmid (sc-437275), UltraCruz transfection reagent (sc-395739), and transfection medium (sc-108062) were from Santa Cruz Biotechnology, Santa Cruz, CA, USA.

Techniques: Transfection, CRISPR, Activation Assay, Incubation, Real-time Polymerase Chain Reaction, Western Blot

Journal: Biomolecules

Article Title: CRISPR/dCas9 Transcriptional Activation of Endogenous Apolipoprotein AI and Paraoxonase 1 in Enterocytes Alleviates Endothelial Cell Dysfunction

doi: 10.3390/biom11121769

Figure Lengend Snippet: Sequence of primers used for Real-Time PCR analysis.

Article Snippet: ApoAI CRISPR activation plasmid (sc-400499-ACT), PON1 CRISPR activation plasmid (sc-402701-ACT), control CRISPR activation plasmid (sc-437275), UltraCruz transfection reagent (sc-395739), and transfection medium (sc-108062) were from Santa Cruz Biotechnology, Santa Cruz, CA, USA.

Techniques: Sequencing, Real-time Polymerase Chain Reaction

Journal: Biomolecules

Article Title: CRISPR/dCas9 Transcriptional Activation of Endogenous Apolipoprotein AI and Paraoxonase 1 in Enterocytes Alleviates Endothelial Cell Dysfunction

doi: 10.3390/biom11121769

Figure Lengend Snippet: Transcriptional activation of ApoAI and PON1 by CRISPR/dCas9 technology in Caco-2 cells. ( a , d ) ApoAI and PON1 mRNA; ( b , e ) ApoAI and PON1 protein expression relative to β-actin (representative blot and densitometric analysis); ( c , f ) secreted ApoAI and PON1 in the culture medium relative to total cell protein (representative blots and densitometric analysis). Cp, control CRISPR activation plasmid; ApoAIp, CRISPR activation plasmids for ApoAI; PON1p, CRISPR activation plasmids for PON1; NC, negative control–untransfected Caco-2 cells. All data are expressed as fold change versus Cp and presented as mean ± SEM of 3–5 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. Cp.

Article Snippet: ApoAI CRISPR activation plasmid (sc-400499-ACT), PON1 CRISPR activation plasmid (sc-402701-ACT), control CRISPR activation plasmid (sc-437275), UltraCruz transfection reagent (sc-395739), and transfection medium (sc-108062) were from Santa Cruz Biotechnology, Santa Cruz, CA, USA.

Techniques: Activation Assay, CRISPR, Expressing, Control, Plasmid Preparation, Negative Control

Journal: Biomolecules

Article Title: CRISPR/dCas9 Transcriptional Activation of Endogenous Apolipoprotein AI and Paraoxonase 1 in Enterocytes Alleviates Endothelial Cell Dysfunction

doi: 10.3390/biom11121769

Figure Lengend Snippet: Overexpression of ApoAI and PON1 determines the increase of lipid transporters protein expression in Caco-2 cells. ( a ) ABCA1, ( b ) SR-BI, and ( c ) ABCG8 protein expression relative to β-actin (representative blot and densitometric analysis). Cp, control CRISPR activation plasmid; ApoAIp, CRISPR activation plasmids for ApoAI; PON1p, CRISPR activation plasmids for PON1; NC, negative control–untransfected Caco-2 cells. All data are expressed as fold change versus Cp and presented as mean ± SEM of 3 independent experiments. * p < 0.05, ** p < 0.01 vs. Cp.

Article Snippet: ApoAI CRISPR activation plasmid (sc-400499-ACT), PON1 CRISPR activation plasmid (sc-402701-ACT), control CRISPR activation plasmid (sc-437275), UltraCruz transfection reagent (sc-395739), and transfection medium (sc-108062) were from Santa Cruz Biotechnology, Santa Cruz, CA, USA.

Techniques: Over Expression, Expressing, Control, CRISPR, Activation Assay, Plasmid Preparation, Negative Control

Journal: Biomolecules

Article Title: CRISPR/dCas9 Transcriptional Activation of Endogenous Apolipoprotein AI and Paraoxonase 1 in Enterocytes Alleviates Endothelial Cell Dysfunction

doi: 10.3390/biom11121769

Figure Lengend Snippet: Overexpression of ApoAI and PON1 increases the protein expression of transcription factors involved in lipid transporters regulation in Caco-2 cells. ( a ) PPARγ, ( b ) LXR, and ( c ) SIRT1 protein expression relative to β-actin (representative blot and densitometric analysis). Cp, control CRISPR activation plasmid; ApoAIp, CRISPR activation plasmids for ApoAI; PON1p, CRISPR activation plasmids for PON1; NC, negative control–untransfected Caco-2 cells. All data are expressed as fold change versus Cp and presented as mean ± SEM of 3 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. Cp.

Article Snippet: ApoAI CRISPR activation plasmid (sc-400499-ACT), PON1 CRISPR activation plasmid (sc-402701-ACT), control CRISPR activation plasmid (sc-437275), UltraCruz transfection reagent (sc-395739), and transfection medium (sc-108062) were from Santa Cruz Biotechnology, Santa Cruz, CA, USA.

Techniques: Over Expression, Expressing, Control, CRISPR, Activation Assay, Plasmid Preparation, Negative Control

Journal: Biomolecules

Article Title: CRISPR/dCas9 Transcriptional Activation of Endogenous Apolipoprotein AI and Paraoxonase 1 in Enterocytes Alleviates Endothelial Cell Dysfunction

doi: 10.3390/biom11121769

Figure Lengend Snippet: Conditioned media from Caco-2 overexpressing ApoAI and PON1 decrease inflammation in TNF-α activated EC. ( a ) TNFR1, ( b ) p22phox, and ( c ) MCP-1 protein expression relative to β-actin (representative blot and densitometric analysis). ( d ) Secreted MCP-1 in the culture medium relative to total cell protein (representative blots and densitometric analysis). Cp, control CRISPR activation plasmid; ApoAIp, CRISPR activation plasmids for ApoAI; PON1p, CRISPR activation plasmids for PON1; CM, conditioned media; NC, negative control–EC incubated with CM from untransfected Caco-2 cells. All data are expressed as fold change versus Cp and presented as mean ± SEM of 3 independent experiments. # p < 0.05, ### p < 0.001 vs. NC; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. Cp_CM.

Article Snippet: ApoAI CRISPR activation plasmid (sc-400499-ACT), PON1 CRISPR activation plasmid (sc-402701-ACT), control CRISPR activation plasmid (sc-437275), UltraCruz transfection reagent (sc-395739), and transfection medium (sc-108062) were from Santa Cruz Biotechnology, Santa Cruz, CA, USA.

Techniques: Expressing, Control, CRISPR, Activation Assay, Plasmid Preparation, Negative Control, Incubation

Journal: Biomolecules

Article Title: CRISPR/dCas9 Transcriptional Activation of Endogenous Apolipoprotein AI and Paraoxonase 1 in Enterocytes Alleviates Endothelial Cell Dysfunction

doi: 10.3390/biom11121769

Figure Lengend Snippet: Schematic representation of the benefic effects induced by the transcriptional activation of the endogenous apoAI or PON1 in enterocytes. Transfection of Caco-2 cells with CRISPR/dCas9 activation plasmids for apoAI or PON1 increases their gene and protein expression, as well as their secretion in the culture medium. Overexpressed apoAI and PON1 exert a positive feedback regulation on their transcription factors PPARγ, LXRs, and SIRT1. In turn, these transcription factors upregulate the lipid transporters ABCA1, ABCG8, and SR-BI which are known to be anti-atherogenic. The apoAI and PON1 secreted by the transfected enterocytes in the culture medium are able to attenuate the TNFα-induced inflammatory and oxidative stress in EC, by decreasing TNFR1, MCP-1, and p22phox levels.

Article Snippet: ApoAI CRISPR activation plasmid (sc-400499-ACT), PON1 CRISPR activation plasmid (sc-402701-ACT), control CRISPR activation plasmid (sc-437275), UltraCruz transfection reagent (sc-395739), and transfection medium (sc-108062) were from Santa Cruz Biotechnology, Santa Cruz, CA, USA.

Techniques: Activation Assay, Transfection, CRISPR, Expressing

Journal: Lipids in Health and Disease

Article Title: The mechanism of apoliprotein A1 down-regulated by Hepatitis B virus

doi: 10.1186/s12944-016-0232-5

Figure Lengend Snippet: ApoA1 expression was significantly decreased in CHB patients. a plasma ApoA1 levels were performed by ELISA in 250 CHB patients corresponding 50 healthy control. b and c ApoA1 protein levels in live tissue sections were detected by Western blot wherase ApoA1 mRNA levels from 200 CHB patients and 50 healthy controls were analyzed by RT-PCR. Results of the Real-time PCR were normalized to an endogenous control GAPDH. The ApoA1 mRNA levels in healthy controls were arbitrarily set as 1.0. Error bars are means ± standard deviation (SD). Data are presented as the mean ± SD from three independent experiments. * p < 0.05 and ** P < 0.01 compared with mock

Article Snippet: SuperscriptTM RT reagent kit (DRR037A, Takara BioInc., Shiga, Japan); ApoA1 specific siRNA and non-specific control (sc-63361, sc-37007, Santa Cruz Biotechnology); the rabbit anti-human ApoA1 (sc-30089, Santa Cruz Biotechnology); the mouse anti-human actin and the horseradish peroxidase-conjugated secondary antibodies (Zhongshan Goldenbridge Biotechnology, China); and the ECL-Plus chemiluminescence system (Applygen Technologies, Beijing, China).

Techniques: Expressing, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Control, Western Blot, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: Lipids in Health and Disease

Article Title: The mechanism of apoliprotein A1 down-regulated by Hepatitis B virus

doi: 10.1186/s12944-016-0232-5

Figure Lengend Snippet: Suppression of ApoA1 expression by HBV. a and b ApoA1 mRNA and protein levels were detected by RT-PCR and Western blot in HepG2.2.15 corresponding HepG2 cell lines. c and d HepG2 cells were transfected with 2 μg pHBV1.3 plasmid or 2 μg pCDNA3.1 as control, ApoA1 mRNA and protein levels were detected at 48 h after transfection. Data are presented as the mean ± SD from three independent experiments. * p < 0.05 and ** P < 0.01 compared with mock

Article Snippet: SuperscriptTM RT reagent kit (DRR037A, Takara BioInc., Shiga, Japan); ApoA1 specific siRNA and non-specific control (sc-63361, sc-37007, Santa Cruz Biotechnology); the rabbit anti-human ApoA1 (sc-30089, Santa Cruz Biotechnology); the mouse anti-human actin and the horseradish peroxidase-conjugated secondary antibodies (Zhongshan Goldenbridge Biotechnology, China); and the ECL-Plus chemiluminescence system (Applygen Technologies, Beijing, China).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection, Plasmid Preparation, Control

Journal: Lipids in Health and Disease

Article Title: The mechanism of apoliprotein A1 down-regulated by Hepatitis B virus

doi: 10.1186/s12944-016-0232-5

Figure Lengend Snippet: ApoA1 expression was suppressed by DNA methyltransferase inhibitor 5-aza-dC. a and b 5 CpG islands including two methylation CpG status in ApoA1 promotor were listed ( a ), the detection results of the 5 CpG islands methylation status were shown ( b ). HepG2.2.15 cells treated with 5 μM 5-aza-dC 48 h, ApoA1 mRNA and protein levels were detected by RT-PCR and Western blot respectively ( c and d ). Secretion of HBsAg and HBV particles in the supernatant were detected by ELISA and RT- PCR respectivly ( e and f ). Data are presented as the mean ± SD from three independent experiments. * p < 0.05 and ** P < 0.01 compared with mock

Article Snippet: SuperscriptTM RT reagent kit (DRR037A, Takara BioInc., Shiga, Japan); ApoA1 specific siRNA and non-specific control (sc-63361, sc-37007, Santa Cruz Biotechnology); the rabbit anti-human ApoA1 (sc-30089, Santa Cruz Biotechnology); the mouse anti-human actin and the horseradish peroxidase-conjugated secondary antibodies (Zhongshan Goldenbridge Biotechnology, China); and the ECL-Plus chemiluminescence system (Applygen Technologies, Beijing, China).

Techniques: Expressing, Methylation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Lipids in Health and Disease

Article Title: The mechanism of apoliprotein A1 down-regulated by Hepatitis B virus

doi: 10.1186/s12944-016-0232-5

Figure Lengend Snippet: Decreased HBV expression with 5-aza-dC treatment via up-regulation of ApoA1 expression. a ApoA1 overexpression inversely suppressed HBV expression. HepG2 cells were cotransfected with 1 μg pApoA1 plasmid and 1 μg pHBV1.3. Sectetion of HBsAg and HBeAg were analyzed by ELISA at 48 h after transfection. b The inhibitory effect of 5-aza-dC on HBV expression was completely abolished by blocking 5-aza-dC-induced up-regulation of ApoA1 using RNAi. HepG2.2.15 cells were treated with 5 μM 5-aza-dC plus 50 μM ApoA1 siRNA or negative control, expression of HBsAg and HBeAg in the supernatant were analyzed by ELISA at 48 h. Data are presented as the mean ± SD from three independent experiments. * p < 0.05 and ** P < 0.01 compared with mock

Article Snippet: SuperscriptTM RT reagent kit (DRR037A, Takara BioInc., Shiga, Japan); ApoA1 specific siRNA and non-specific control (sc-63361, sc-37007, Santa Cruz Biotechnology); the rabbit anti-human ApoA1 (sc-30089, Santa Cruz Biotechnology); the mouse anti-human actin and the horseradish peroxidase-conjugated secondary antibodies (Zhongshan Goldenbridge Biotechnology, China); and the ECL-Plus chemiluminescence system (Applygen Technologies, Beijing, China).

Techniques: Expressing, Over Expression, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Transfection, Blocking Assay, Negative Control

Journal: Frontiers in Physiology

Article Title: β-Cyclodextrins Decrease Cholesterol Release and ABC-Associated Transporter Expression in Smooth Muscle Cells and Aortic Endothelial Cells

doi: 10.3389/fphys.2016.00185

Figure Lengend Snippet: Cholesterol efflux from ABAE and SMCs . Cells were first labeled with [ 3 H]-cholesterol (0.5 μCi/mL) for 36 h at 37°C and then equilibrated for 24 h in DMEM/0.05% BSA at 37°C. Cellular [ 3 H]-cholesterol loads in DPM per μg of proteins were compared between ABAE and SMCs (A) . Data represent the mean ± SEM (in DPM/μg of proteins), with n = 6 from two sets of experiments. Paired- t -test was performed, *** p < 0.001 when compared with ABAE condition. [ 3 H]-cholesterol released from both cell types was then measured 6 h after medium supplementation with either BSA (control condition) or cholesterol acceptors [ApoA-I (20 μg/mL) or HDL (50 μg/mL)] at 37°C (B) . Data represent the mean ± SEM (in DPM/μg of proteins), with n = 6 from two sets of experiments. One-way ANOVA test followed by Bonferroni correction was performed in which *** p < 0.001 refers to the control condition and ### p < 0.001 refers to the matched HDL condition in ABAE.

Article Snippet: ApoA-I and HDL were purchased from PROSCI Incorporated (Poway, CA, USA).

Techniques: Labeling, Control

Journal: Frontiers in Physiology

Article Title: β-Cyclodextrins Decrease Cholesterol Release and ABC-Associated Transporter Expression in Smooth Muscle Cells and Aortic Endothelial Cells

doi: 10.3389/fphys.2016.00185

Figure Lengend Snippet: Cholesterol release from ABAE (A,C) and from SMCs (B,D) upon CD-treatment . Cells were first labeled with [ 3 H]-cholesterol (0.5 μCi/mL) for 36 h at 37°C and then equilibrated in DMEM/0.05% BSA for 24 h at 37°C. [ 3 H]-cholesterol released in the medium from both cell types was then measured 6 h after medium supplementation with cholesterol acceptors [ApoA-I (20 μg/mL) or HDL (50 μg/mL)] at 37°C. The radioactivity contained in the culture medium, that corresponds to the cholesterol released from cells was measured. Data are expressed as the mean ± SEM (in DPM/μg of proteins, n = 6 from two sets of experiments). Statistical analysis: a one-way ANOVA followed by Dunnett's test for multiple comparisons in which * p < 0.05; *** p < 0.001 compared with the control condition (open bars) without any CD treatment.

Article Snippet: ApoA-I and HDL were purchased from PROSCI Incorporated (Poway, CA, USA).

Techniques: Labeling, Radioactivity, Control

Journal: Frontiers in Physiology

Article Title: β-Cyclodextrins Decrease Cholesterol Release and ABC-Associated Transporter Expression in Smooth Muscle Cells and Aortic Endothelial Cells

doi: 10.3389/fphys.2016.00185

Figure Lengend Snippet: Effect of 10 μM T0901317 on cholesterol efflux to ApoA-I and HDL in ABAE (A) and SMCs (B) treated or nor with Rameβ . Cells were first labeled with [ 3 H]-cholesterol as previously described and incubated in the presence of 1 mM Rameβ, 10 μM T0901317 or both during 24 h. Then, [ 3 H]-cholesterol released in the medium from both cell types was measured 6 h after medium supplementation with either ApoA-I (20 μg/mL) or HDL (50 μg/mL). Data are expressed as the mean ± SEM (in DPM/μg of proteins, n = 6 from two sets of experiments). Control condition (without any treatment) is used as the reference level and set to 100%. Statistical analysis: a one-way ANOVA followed by Dunnett's test for multiple comparisons in which * p < 0.05; ** p < 0.01; *** p < 0.001 compared with the untreated condition (Control condition, open bars) and in which ## p < 0.01 when Rameβ + T0901317 condition was compared with Rameβ condition.

Article Snippet: ApoA-I and HDL were purchased from PROSCI Incorporated (Poway, CA, USA).

Techniques: Labeling, Incubation, Control