Journal: Bioscience Reports

Article Title: Melatonin targets ferroptosis through bimodal alteration of redox environment and cellular pathways in NAFLD model

doi: 10.1042/BSR20230128

Figure Lengend Snippet: ( A ) Oil Red O staining of lipid droplets in both control and FFA-treated cells. ( B ) Bar graph of GSH and ( C ) MDA content signifying changes in oxidative environment of cells. ( D ) Representative Western blot, and ( E,F ) bar graphs show the quantification of SLC7A11/β-Actin and GPX4/β-Actin, respectively. Panels ( G,H ) represent viability of HepG2 cells at different concentrations of MLT and SAS. Panel ( I ) represents total iron content in FFA/MLT-treated HepG2 cells. Data are represented as the mean percentage ± SEM ( n =3); **** P <0.0001, *** P <0.001, ** P <0.01, *P <0.05; ns, non-significant.

Article Snippet: NRF2 gene expression was knocked down by transfecting 1 × 10 6 HepG2 cells with siRNA-NRF2 (sc-37030, Santa Cruz, U.S.A.) or siRNA control.

Techniques: Staining, Control, Western Blot

Journal: Bioscience Reports

Article Title: Melatonin targets ferroptosis through bimodal alteration of redox environment and cellular pathways in NAFLD model

doi: 10.1042/BSR20230128

Figure Lengend Snippet: Qualitative and quantitative analysis of intracellular oxidative status and involvement of Nrf2/HO-1 pathway in NAFLD-related ferroptosis. ( A ) Flow cytometric analysis of ROS generation in different treatment groups using DCFDA, and ( B ) bar graph represents mean fluorescence unit of ROS levels in different groups. Panels ( C–E ) represent MDA, SOD and GSH content in different treated groups. ( F ) Western blot analysis of Nrf2, HO-1 and Keap-1. ( G ) Bar graphs show the quantification of Nrf2/β-Actin, HO-1/β-Actin and Keap-1/β-Actin, respectively. ( H ) Representative immunofluorescence images shown Nrf2 and HO-1 expression in HepG2 cells depending on different treatments; scale bar: 50 µm. Data are represented as the mean percentage ± SEM ( n =3); **** P <0.0001, *** P <0.001, ** P <0.01, * P <0.05; ns, non-significant.

Article Snippet: NRF2 gene expression was knocked down by transfecting 1 × 10 6 HepG2 cells with siRNA-NRF2 (sc-37030, Santa Cruz, U.S.A.) or siRNA control.

Techniques: Fluorescence, Western Blot, Immunofluorescence, Expressing

Journal: Bioscience Reports

Article Title: Melatonin targets ferroptosis through bimodal alteration of redox environment and cellular pathways in NAFLD model

doi: 10.1042/BSR20230128

Figure Lengend Snippet: SLC7A11 and GPX4 expression was verified using Nrf2 Si-RNA transfection. ( A ) Western blot analysis of Nrf2 in control and SiRNA-treated groups. ( B ) Bar graphs show the quantification of Nrf2/β-Actin. ( C ) Western blot analysis of SLC7A11 and GPX4. ( D ) Bar graphs show the quantification of SLC7A11/β-Actin and GPX4/β-Actin, respectively. ( E ) Immunofluorescence images of HepG2 cells of different treated groups using BODIPY; scale bar: 50 µm. Data are represented as the mean percentage ± SEM ( n =3); **** P <0.0001, ***P <0.001, ** P <0.01, * P <0.05; ns, non-significant.

Article Snippet: NRF2 gene expression was knocked down by transfecting 1 × 10 6 HepG2 cells with siRNA-NRF2 (sc-37030, Santa Cruz, U.S.A.) or siRNA control.

Techniques: Expressing, Transfection, Western Blot, Control, Immunofluorescence

Journal: Bioscience Reports

Article Title: Melatonin targets ferroptosis through bimodal alteration of redox environment and cellular pathways in NAFLD model

doi: 10.1042/BSR20230128

Figure Lengend Snippet: Analysis of lipid ROS and potential lipogenic markers. ( A ) BODIPY staining of HepG2 cells treated with FFA and different doses of MLT. ( B ) Western blot analyses of pAMPKα, total AMPKα, PPARα, PPARγ, SREBP1c and FAS are shown. ( C ) Bar graphs show the quantification of total AMPKα/β-Actin, pAMPKα/Total AMPKα, PPARα/β-Actin, PPARγ/β-Actin, FAS/β-Actin and SREBP1c/β-Actin, respectively. ( D ) Immunofluorescence images of expression of pAMPKα and SREBP1c; scale bar: 50 µm. Data are represented as the mean percentage ± SEM ( n =3); **** P <0.0001, *** P <0.001, ** P <0.01, * P <0.05; ns, non-significant.

Article Snippet: NRF2 gene expression was knocked down by transfecting 1 × 10 6 HepG2 cells with siRNA-NRF2 (sc-37030, Santa Cruz, U.S.A.) or siRNA control.

Techniques: Staining, Western Blot, Immunofluorescence, Expressing

Journal: Bioscience Reports

Article Title: Melatonin targets ferroptosis through bimodal alteration of redox environment and cellular pathways in NAFLD model

doi: 10.1042/bsr20230128

Figure Lengend Snippet: Figure 1. NAFLD-linked ferroptosis was induced by FFA in HepG2 cells

Article Snippet: After incubation with secondary antibodies for 1–2 h at ambient room temperature, then the detection was done using NBT/BCIP chromogenic substrate and the relative expression of the respective proteins were analysed using ImageJ software (U.S.A.). siRNA transfection NRF2 gene expression was knocked down by transfecting 1 × 106 HepG2 cells with siRNA-NRF2 (sc-37030, Santa Cruz, U.S.A.) or siRNA control.

Techniques:

Journal: Bioscience Reports

Article Title: Melatonin targets ferroptosis through bimodal alteration of redox environment and cellular pathways in NAFLD model

doi: 10.1042/bsr20230128

Figure Lengend Snippet: Figure 3. Melatonin recovers HepG2 cells from FFA-induced ferroptosis via modifying Nrf2/HO-1 mediated signaling Qualitative and quantitative analysis of intracellular oxidative status and involvement of Nrf2/HO-1 pathway in NAFLD-related fer- roptosis. (A) Flow cytometric analysis of ROS generation in different treatment groups using DCFDA, and (B) bar graph represents

Article Snippet: After incubation with secondary antibodies for 1–2 h at ambient room temperature, then the detection was done using NBT/BCIP chromogenic substrate and the relative expression of the respective proteins were analysed using ImageJ software (U.S.A.). siRNA transfection NRF2 gene expression was knocked down by transfecting 1 × 106 HepG2 cells with siRNA-NRF2 (sc-37030, Santa Cruz, U.S.A.) or siRNA control.

Techniques:

Journal: Bioscience Reports

Article Title: Melatonin targets ferroptosis through bimodal alteration of redox environment and cellular pathways in NAFLD model

doi: 10.1042/bsr20230128

Figure Lengend Snippet: Figure 4. Melatonin recovers HepG2 cells from FFA-induced ferroptosis via modifying Nrf2/HO-1-mediated signaling

Article Snippet: After incubation with secondary antibodies for 1–2 h at ambient room temperature, then the detection was done using NBT/BCIP chromogenic substrate and the relative expression of the respective proteins were analysed using ImageJ software (U.S.A.). siRNA transfection NRF2 gene expression was knocked down by transfecting 1 × 106 HepG2 cells with siRNA-NRF2 (sc-37030, Santa Cruz, U.S.A.) or siRNA control.

Techniques:

Journal: Bioscience Reports

Article Title: Melatonin targets ferroptosis through bimodal alteration of redox environment and cellular pathways in NAFLD model

doi: 10.1042/bsr20230128

Figure Lengend Snippet: Figure 6. Melatonin inhibited metabolic markers associated with lipogenesis in vitro Analysis of lipid ROS and potential lipogenic markers. (A) BODIPY staining of HepG2 cells treated with FFA and different doses of MLT. (B) Western blot analyses of pAMPKα, total AMPKα, PPARα, PPARγ, SREBP1c and FAS are shown. (C) Bar

Article Snippet: After incubation with secondary antibodies for 1–2 h at ambient room temperature, then the detection was done using NBT/BCIP chromogenic substrate and the relative expression of the respective proteins were analysed using ImageJ software (U.S.A.). siRNA transfection NRF2 gene expression was knocked down by transfecting 1 × 106 HepG2 cells with siRNA-NRF2 (sc-37030, Santa Cruz, U.S.A.) or siRNA control.

Techniques: In Vitro, Staining, Western Blot

Journal: Antioxidants

Article Title: Cell-Based Antioxidant Properties and Synergistic Effects of Natural Plant and Algal Extracts Pre and Post Intestinal Barrier Transport

doi: 10.3390/antiox11030565

Figure Lengend Snippet: Comparison of AOP1 EC 50 s evaluated in Caco-2 and HepG2 cells for the 12 natural extracts selected for the intestinal barrier transport study. Extracts were selected according to the best EC 50 s obtained with AOP1 assay. In order to increase the diversity of tested extracts, some Vitis vinifera extracts were removed from the list because of redundancy with each other. ND = not determined (no full effect observed).

Article Snippet: The HepG2/Nrf2 cells were treated with conditions for 17 h; then, they were treated with a mix (BPS Bioscience, USA) comprising cell lysis solution and luciferin (substrate of luciferase) for 40 min. Luminescence was read on a Varioskan Flash Spectral Scanning Multimode Reader.

Techniques:

Journal: Antioxidants

Article Title: Cell-Based Antioxidant Properties and Synergistic Effects of Natural Plant and Algal Extracts Pre and Post Intestinal Barrier Transport

doi: 10.3390/antiox11030565

Figure Lengend Snippet: Indirect antioxidant ARE/Nrf2 assay data obtained for Syzygium aromaticum extract (given as an example) in HepG2/Nrf2 cells. All other extracts were subjected to the same protocol and data analyses. ( A ) Assay of increasing concentrations showing the typical bell-shaped profile (see text for explanation). Sulforaphane 25 µM was used as positive control; ( B ) dose–response and sigmoid fit curve used for Efficacy Concentration (EC) estimations.

Article Snippet: The HepG2/Nrf2 cells were treated with conditions for 17 h; then, they were treated with a mix (BPS Bioscience, USA) comprising cell lysis solution and luciferin (substrate of luciferase) for 40 min. Luminescence was read on a Varioskan Flash Spectral Scanning Multimode Reader.

Techniques: Positive Control, Concentration Assay

Journal: Antioxidants

Article Title: Cell-Based Antioxidant Properties and Synergistic Effects of Natural Plant and Algal Extracts Pre and Post Intestinal Barrier Transport

doi: 10.3390/antiox11030565

Figure Lengend Snippet: ARE/Nrf2 EC 50 s established in HepG2 cells for the 12 natural extracts selected for the Caco-2 intestinal barrier transport study. ND = not determined; R 2 = determination coefficient.

Article Snippet: The HepG2/Nrf2 cells were treated with conditions for 17 h; then, they were treated with a mix (BPS Bioscience, USA) comprising cell lysis solution and luciferin (substrate of luciferase) for 40 min. Luminescence was read on a Varioskan Flash Spectral Scanning Multimode Reader.

Techniques:

Journal: Antioxidants

Article Title: Cell-Based Antioxidant Properties and Synergistic Effects of Natural Plant and Algal Extracts Pre and Post Intestinal Barrier Transport

doi: 10.3390/antiox11030565

Figure Lengend Snippet: Effect of post-intestinal barrier transport compartments on ARE/NRF2 pathway activation in HepG2 cells. Results are expressed as fold increase compared to constitutive medium ARE/Nrf2 gene expression. Values represent means of quadruplicates obtained from two independent intestinal barrier transport experiments. Sulforaphane 25 µM data is provided as positive control; *** p < 0.005.

Article Snippet: The HepG2/Nrf2 cells were treated with conditions for 17 h; then, they were treated with a mix (BPS Bioscience, USA) comprising cell lysis solution and luciferin (substrate of luciferase) for 40 min. Luminescence was read on a Varioskan Flash Spectral Scanning Multimode Reader.

Techniques: Activation Assay, Expressing, Positive Control

Journal: Antioxidants

Article Title: Cell-Based Antioxidant Properties and Synergistic Effects of Natural Plant and Algal Extracts Pre and Post Intestinal Barrier Transport

doi: 10.3390/antiox11030565

Figure Lengend Snippet: Effect of combinations of post-intestinal barrier compartments on ARE/NRF2 pathway in HepG2 cells. Results are expressed as ARE/Nrf2 gene expression fold increase compared to constitutive medium. Values represent means of pentaplicates for all 50/50 combinations and of triplicates for all others. All data were normalized to constitutive medium values; *** p < 0.001; ** p < 0.01.

Article Snippet: The HepG2/Nrf2 cells were treated with conditions for 17 h; then, they were treated with a mix (BPS Bioscience, USA) comprising cell lysis solution and luciferin (substrate of luciferase) for 40 min. Luminescence was read on a Varioskan Flash Spectral Scanning Multimode Reader.

Techniques: Expressing

Journal: Antioxidants

Article Title: Cell-Based Antioxidant Properties and Synergistic Effects of Natural Plant and Algal Extracts Pre and Post Intestinal Barrier Transport

doi: 10.3390/antiox11030565

Figure Lengend Snippet: Synergistic effects on ARE/Nrf2 gene expression of post-intestinal barrier compartment combinations. Combin. FI = gene expression fold increase (FI) measure for the combination; S indep. FI = sum of the increase in gene expression fold increase (FI) measures of each of the two extracts taken independently as depicted in Figure 6 ; Var. (%) = variation in percentage of the two values. A positive percentage value indicates a synergistic effect.

Article Snippet: The HepG2/Nrf2 cells were treated with conditions for 17 h; then, they were treated with a mix (BPS Bioscience, USA) comprising cell lysis solution and luciferin (substrate of luciferase) for 40 min. Luminescence was read on a Varioskan Flash Spectral Scanning Multimode Reader.

Techniques: Expressing

Journal: Antioxidants

Article Title: Cell-Based Antioxidant Properties and Synergistic Effects of Natural Plant and Algal Extracts Pre and Post Intestinal Barrier Transport

doi: 10.3390/antiox11030565

Figure Lengend Snippet: Indirect antioxidant ARE/Nrf2 assay data obtained for Syzygium aromaticum extract (given as an example) in HepG2/Nrf2 cells. All other extracts were subjected to the same protocol and data analyses. ( A ) Assay of increasing concentrations showing the typical bell-shaped profile (see text for explanation). Sulforaphane 25 µM was used as positive control; ( B ) dose–response and sigmoid fit curve used for Efficacy Concentration (EC) estimations.

Article Snippet: A HepG2 ARE reporter cell line (Nrf2 antioxidant pathway) was purchased from BPS Bioscience (catalogue number 60513).

Techniques: Positive Control, Concentration Assay

Journal: Antioxidants

Article Title: Cell-Based Antioxidant Properties and Synergistic Effects of Natural Plant and Algal Extracts Pre and Post Intestinal Barrier Transport

doi: 10.3390/antiox11030565

Figure Lengend Snippet: ARE/Nrf2 EC 50 s established in HepG2 cells for the 12 natural extracts selected for the Caco-2 intestinal barrier transport study. ND = not determined; R 2 = determination coefficient.

Article Snippet: A HepG2 ARE reporter cell line (Nrf2 antioxidant pathway) was purchased from BPS Bioscience (catalogue number 60513).

Techniques:

Journal: Antioxidants

Article Title: Cell-Based Antioxidant Properties and Synergistic Effects of Natural Plant and Algal Extracts Pre and Post Intestinal Barrier Transport

doi: 10.3390/antiox11030565

Figure Lengend Snippet: Effect of post-intestinal barrier transport compartments on ARE/NRF2 pathway activation in HepG2 cells. Results are expressed as fold increase compared to constitutive medium ARE/Nrf2 gene expression. Values represent means of quadruplicates obtained from two independent intestinal barrier transport experiments. Sulforaphane 25 µM data is provided as positive control; *** p < 0.005.

Article Snippet: A HepG2 ARE reporter cell line (Nrf2 antioxidant pathway) was purchased from BPS Bioscience (catalogue number 60513).

Techniques: Activation Assay, Expressing, Positive Control

Journal: Antioxidants

Article Title: Cell-Based Antioxidant Properties and Synergistic Effects of Natural Plant and Algal Extracts Pre and Post Intestinal Barrier Transport

doi: 10.3390/antiox11030565

Figure Lengend Snippet: Effect of combinations of post-intestinal barrier compartments on ARE/NRF2 pathway in HepG2 cells. Results are expressed as ARE/Nrf2 gene expression fold increase compared to constitutive medium. Values represent means of pentaplicates for all 50/50 combinations and of triplicates for all others. All data were normalized to constitutive medium values; *** p < 0.001; ** p < 0.01.

Article Snippet: A HepG2 ARE reporter cell line (Nrf2 antioxidant pathway) was purchased from BPS Bioscience (catalogue number 60513).

Techniques: Expressing

Journal: Antioxidants

Article Title: Cell-Based Antioxidant Properties and Synergistic Effects of Natural Plant and Algal Extracts Pre and Post Intestinal Barrier Transport

doi: 10.3390/antiox11030565

Figure Lengend Snippet: Synergistic effects on ARE/Nrf2 gene expression of post-intestinal barrier compartment combinations. Combin. FI = gene expression fold increase (FI) measure for the combination; S indep. FI = sum of the increase in gene expression fold increase (FI) measures of each of the two extracts taken independently as depicted in Figure 6 ; Var. (%) = variation in percentage of the two values. A positive percentage value indicates a synergistic effect.

Article Snippet: A HepG2 ARE reporter cell line (Nrf2 antioxidant pathway) was purchased from BPS Bioscience (catalogue number 60513).

Techniques: Expressing