Journal: Journal of Natural Medicines

Article Title: Inhibitory effect of trans -tiliroside on very low-density lipoprotein secretion in HepG2 cells and mouse liver

doi: 10.1007/s11418-023-01756-0

Figure Lengend Snippet: Effects of compounds 1 and 2 on apoB-100 secretion in HepG2 cells. HepG2 cells were treated with 1 or 2 in 20 mM mevalonate-supplemented DMEM (FBS-and phenol red-free) for 24 h. The apoB-100 content in medium were determined by ELISA using commercial kit. Each bar represents the mean ± S.E.M. ( n = 4). Significantly different from the control, * p < 0.05, ** p < 0.01 (Dunnett’s test)

Article Snippet: Determination of apoB-100 in medium or mouse plasma was performed using commercial sandwich ELISA kits and an APOB ELISA Kit (Proteintech Group, Inc., Rosemont, IL, USA) for medium, and a Mouse Apo B ELISA Kit (Abcam plc, Cambridge, UK) for mouse plasma, according to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Control

Journal: Journal of Natural Medicines

Article Title: Inhibitory effect of trans -tiliroside on very low-density lipoprotein secretion in HepG2 cells and mouse liver

doi: 10.1007/s11418-023-01756-0

Figure Lengend Snippet: Effects of compounds 1 and 2 on plasma TG and apoB-100 levels in Triton WR-1339-treated mice. The mice were orally administered the sample solution for 7 days. On the sixth day, the mice were deprived of food after sample administration. After 18 h, Triton WR-1339 (400 mg/kg) was injected (i.p.) 1 h after the final administration of the sample. Blood samples were collected at 0, 1, 2, and 4 h after Triton WR-1339 injection. Plasma TG concentration ( a ) was measured calorimetrically, and the area under the curve (AUC 0–4 ) ( b ) was calculated using the trapezoidal method. Plasma apoB-100 concentration at 4 h after Triton WR-1339 injection ( c ) was measured using a commercial ELISA kit. Each bar represents the mean ± S.E.M. ( n = 10–11). Significantly different from the control, * p < 0.05, ** p < 0.01 (Dunnett’s test)

Article Snippet: Determination of apoB-100 in medium or mouse plasma was performed using commercial sandwich ELISA kits and an APOB ELISA Kit (Proteintech Group, Inc., Rosemont, IL, USA) for medium, and a Mouse Apo B ELISA Kit (Abcam plc, Cambridge, UK) for mouse plasma, according to the manufacturer’s instructions.

Techniques: Clinical Proteomics, Injection, Concentration Assay, Enzyme-linked Immunosorbent Assay, 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: 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 apoB levels in the collected media were determined with a human apoB ELISA development kit (MABTECH, Inc; catalog no.: 3715-1H-6) in 96-well ELISA plates (Thermo Fisher Scientific; catalog no.: 07-200-640) with 3,3′,5,5′ tetramethylbenzidine substrate (Thermo Fisher Scientific; catalog no.: 4041).

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 apoB levels in the collected media were determined with a human apoB ELISA development kit (MABTECH, Inc; catalog no.: 3715-1H-6) in 96-well ELISA plates (Thermo Fisher Scientific; catalog no.: 07-200-640) with 3,3′,5,5′ tetramethylbenzidine substrate (Thermo Fisher Scientific; catalog no.: 4041).

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 apoB levels in the collected media were determined with a human apoB ELISA development kit (MABTECH, Inc; catalog no.: 3715-1H-6) in 96-well ELISA plates (Thermo Fisher Scientific; catalog no.: 07-200-640) with 3,3′,5,5′ tetramethylbenzidine substrate (Thermo Fisher Scientific; catalog no.: 4041).

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 apoB levels in the collected media were determined with a human apoB ELISA development kit (MABTECH, Inc; catalog no.: 3715-1H-6) in 96-well ELISA plates (Thermo Fisher Scientific; catalog no.: 07-200-640) with 3,3′,5,5′ tetramethylbenzidine substrate (Thermo Fisher Scientific; catalog no.: 4041).

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 apoB levels in the collected media were determined with a human apoB ELISA development kit (MABTECH, Inc; catalog no.: 3715-1H-6) in 96-well ELISA plates (Thermo Fisher Scientific; catalog no.: 07-200-640) with 3,3′,5,5′ tetramethylbenzidine substrate (Thermo Fisher Scientific; catalog no.: 4041).

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 apoB levels in the collected media were determined with a human apoB ELISA development kit (MABTECH, Inc; catalog no.: 3715-1H-6) in 96-well ELISA plates (Thermo Fisher Scientific; catalog no.: 07-200-640) with 3,3′,5,5′ tetramethylbenzidine substrate (Thermo Fisher Scientific; catalog no.: 4041).

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