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: iScience

Article Title: Selective activation of ABCA1/ApoA1 signaling in the V1 by magnetoelectric stimulation ameliorates depression via regulation of synaptic plasticity

doi: 10.1016/j.isci.2022.104201

Figure Lengend Snippet:

Article Snippet: Mouse ApoA1(Apolipoprotein A1) ELISA Kit , Elabscience , Cat#E-EL-M3016.

Techniques: Virus, Recombinant, Extraction, Staining, TUNEL Assay, Enzyme-linked Immunosorbent Assay, Cholesterol Assay, shRNA, Real-time Polymerase Chain Reaction, Software

Journal: The Journal of Biological Chemistry

Article Title: Novel efficacious microRNA-30c analogs reduce apolipoprotein B secretion in human hepatoma and primary hepatocyte cells

doi: 10.1016/j.jbc.2022.101813

Figure Lengend Snippet: Activity of 2′-OMe modified miR-30c-1 and miR-30c-2 in Huh-7 human hepatoma cells. Huh-7 cells were reverse transfected with 100 nM of 2′-OMe-modified miR-30c analogs complexed with Lipofectamine RNAiMax transfection reagent at a ratio of 3:1. For the control, cells were treated only with Lipofectamine RNAiMax transfection reagent (no mimics). Forty-eight hours after transfection with 2′-OMe-30c-1 and 2′-OMe-30c-2 analogs, media were collected and used to measure apoB and apoA1 concentrations. Data are representative of three independent experiments. Significance was determined at p < 0.05 (∗) with two-tailed t test, and error bars represent mean ± SD ( A – D ). apoA1, apolipoprotein A1; apoB, apolipoprotein B; miR-30c, microRNA-30c.

Article Snippet: The apoA1 levels in the collected media were determined with a human apolipoprotein A-I/ApoA1 DuoSet ELISA kit (R&D Systems; catalog no.: DY3664) in 96-well ELISA plates (Thermo Fisher Scientific; catalog no.: 7-200-640).

Techniques: Activity Assay, Modification, Transfection, Control, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Novel efficacious microRNA-30c analogs reduce apolipoprotein B secretion in human hepatoma and primary hepatocyte cells

doi: 10.1016/j.jbc.2022.101813

Figure Lengend Snippet: Effects of different miR-30c series A analogs on apoB secretion in Huh-7 human hepatoma cells. A , apoB secretion in Huh-7 cells transfected with control (Ctrl) and different miR-30c analogs (100 nM), as described in Table 1 , with Lipofectamine RNAiMax transfection reagent. MiR-30c (30c) was used as a positive control. B , apoA1 secretion in Huh-7 cells transfected with control (Ctrl) and different miR-30c analogs (100 nM) with Lipofectamine RNAiMax transfection reagent. C , apoB secretion in Huh-7 cells transfected with Ctrl and different miR-30c analogs (1000 nM) without Lipofectamine RNAiMax transfection reagent. D , apoA1 secretion in Huh-7 cells transfected with Ctrl and different miR-30c analogs without Lipofectamine RNAiMax transfection reagent. The apoB and apoA1 concentrations were measured by ELISA in the collected media. Data are representative of four independent experiments. Significance was determined at p < 0.0001 (∗∗∗∗) and p < 0.001 (∗∗∗) with one-way ANOVA, and error bars represent mean ± SD ( A – D ). apoA1, apolipoprotein A1; apoB, apolipoprotein B; miR-30c, microRNA-30c.

Article Snippet: The apoA1 levels in the collected media were determined with a human apolipoprotein A-I/ApoA1 DuoSet ELISA kit (R&D Systems; catalog no.: DY3664) in 96-well ELISA plates (Thermo Fisher Scientific; catalog no.: 7-200-640).

Techniques: Transfection, Control, Positive Control, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Biological Chemistry

Article Title: Novel efficacious microRNA-30c analogs reduce apolipoprotein B secretion in human hepatoma and primary hepatocyte cells

doi: 10.1016/j.jbc.2022.101813

Figure Lengend Snippet: Effects of various GalNAc-modified miR-30c analogs on apoB secretion in Huh-7 human hepatoma cells. A , apoB secretion and ( B ) apoA1 secretion in Huh-7 cells transfected with control (Ctrl) and different miR-30c series B analogs ( Table 2 ) with Lipofectamine RNAiMax transfection reagent. MiR-30c (30c) was used as a positive control. C , apoB and ( D ) apoA1 secretion in Huh-7 cells transfected with Ctrl and different miR-30c analogs without Lipofectamine RNAiMax transfection reagent. The apoB and apoA1 concentrations in the media were measured with ELISA. Data are representative of four independent experiments. Significance was determined at p < 0.0001 (∗∗∗∗) and p < 0.001 (∗∗∗) with one-way ANOVA, and error bars represent mean ± SD ( A – D ). apoA1, apolipoprotein A1; apoB, apolipoprotein B; miR-30c, microRNA-30c.

Article Snippet: The apoA1 levels in the collected media were determined with a human apolipoprotein A-I/ApoA1 DuoSet ELISA kit (R&D Systems; catalog no.: DY3664) in 96-well ELISA plates (Thermo Fisher Scientific; catalog no.: 7-200-640).

Techniques: Modification, Transfection, Control, Positive Control, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Biological Chemistry

Article Title: Novel efficacious microRNA-30c analogs reduce apolipoprotein B secretion in human hepatoma and primary hepatocyte cells

doi: 10.1016/j.jbc.2022.101813

Figure Lengend Snippet: Effects of GalNAc-modified B1 and B2 analogs on apoB secretion and MTP activity in Huh-7 human hepatoma cells. ( A ) apoB and ( B ) apoA1 secretion (%) in the culture medium of Huh-7 cells transfected with increasing amounts of miR-30c or B1 or B2 analog without Lipofectamine RNAiMax transfection reagent. Nontransfected cells were used as controls. Data are representative of two independent experiments. C – F , cells were exposed to 250 nM of miR-30c or B1 without transfection reagent. After 48 h, the cells were used to measure ( C ) miR-30c, ( D ) MTP mRNA, ( E ) MTP activity, and ( F ) MTP levels. Data are representative of three independent experiments. Western blot analysis is from one representative experiment. After transfer, the blot was cut into two parts; the top was used to detect MTP, and the bottom was used to detect β-actin as a loading control. Changes in protein levels were quantified using ImageJ. Significance was determined at p < 0.05 (∗) and p < 0.0001 (∗∗∗∗) with two-way ANOVA ( A and B ), one-way ANOVA ( C – E ), and error bars represent mean ± SD. apoB, apolipoprotein B; miR-30c, microRNA-30c; MTP, microsomal triglyceride transfer protein.

Article Snippet: The apoA1 levels in the collected media were determined with a human apolipoprotein A-I/ApoA1 DuoSet ELISA kit (R&D Systems; catalog no.: DY3664) in 96-well ELISA plates (Thermo Fisher Scientific; catalog no.: 7-200-640).

Techniques: Modification, Activity Assay, Transfection, Western Blot, Control

Journal: The Journal of Biological Chemistry

Article Title: Novel efficacious microRNA-30c analogs reduce apolipoprotein B secretion in human hepatoma and primary hepatocyte cells

doi: 10.1016/j.jbc.2022.101813

Figure Lengend Snippet: Effects of various phosphorothioate-linked miR-30c analogs on apoB secretion in Huh-7 human hepatoma cells. ( A ) apoB and ( B ) apoA1 secretion in Huh-7 cells transfected with Ctrl and different synthetic miR-30c series C analogs ( Table 3 ) with Lipofectamine RNAiMax transfection reagent. MiR-30c (30c) was used as a positive control. ( C ) apoB and ( D ) apoA1 secretion in Huh-7 cells transfected with Ctrl and different miR-30c analogs without Lipofectamine RNAiMax transfection reagent. Data are representative of three independent experiments. Relative ( E ) apoB and ( F ) apoA1 secretion (%) in culture medium of Huh-7 cells transfected with increasing amounts of miR-30c or analog without Lipofectamine RNAiMax transfection reagent. Nontransfected cells were used as a control. Data are representative of two independent experiments. Significance was determined at p < 0.0001 (∗∗∗∗), p < 0.001 (∗∗∗), and p < 0.01 (∗∗) with two-way ANOVA ( E and F ), and error bars represent mean ± SD. apoA1, apolipoprotein A1; apoB, apolipoprotein B; miR-30c, microRNA-30c.

Article Snippet: The apoA1 levels in the collected media were determined with a human apolipoprotein A-I/ApoA1 DuoSet ELISA kit (R&D Systems; catalog no.: DY3664) in 96-well ELISA plates (Thermo Fisher Scientific; catalog no.: 7-200-640).

Techniques: Transfection, Positive Control, Control

Journal: The Journal of Biological Chemistry

Article Title: Novel efficacious microRNA-30c analogs reduce apolipoprotein B secretion in human hepatoma and primary hepatocyte cells

doi: 10.1016/j.jbc.2022.101813

Figure Lengend Snippet: Effects of C2 analog on lipid levels in Huh-7 human hepatoma cells and in human primary hepatocytes. A and B , Huh-7 cells were incubated in triplicate with miR-30c or C2 analog (200 nM) without Lipofectamine RNAiMax transfection reagent. After 72 h, media were changed. After 16 h, we collected media to measure ( A ) apoB and ( B ) apoA1 protein levels using ELISA. Cells were used to measure protein levels. Amounts of proteins secreted were normalized to cellular protein. Data are presented as percent of miR-30c-treated cells (control). Data are representative of two independent experiments. C and D , in separate studies, Huh-7 cells were incubated with miR-30c or analog C2 without Lipofectamine RNAiMax transfection reagent as described previously. Cells were incubated with isopropanol to extract lipids and measure ( C ) triglycerides and ( D ) cholesterol levels. Representative data of two independent studies. E and F , human primary hepatocytes were incubated in triplicate with miR-30c or C2 analog (200 nM) without Lipofectamine RNAiMax transfection reagent. Media were used to measure ( E ) apoB and ( F ) apoA1, and cells were used to measure protein levels. G and H , in a separate study, miR-30c-treated or C2 analog–treated human primary hepatocytes were used to measure cellular ( G ) triglyceride and ( H ) cholesterol levels. Significance was determined at p < 0.01 (∗∗) with two-tailed t test, and error bars represent mean ± SD ( A – H ). apoA1, apolipoprotein A1; apoB, apolipoprotein B; miR-30c, microRNA-30c.

Article Snippet: The apoA1 levels in the collected media were determined with a human apolipoprotein A-I/ApoA1 DuoSet ELISA kit (R&D Systems; catalog no.: DY3664) in 96-well ELISA plates (Thermo Fisher Scientific; catalog no.: 7-200-640).

Techniques: Incubation, Transfection, Enzyme-linked Immunosorbent Assay, Control, Two Tailed Test