Journal: Journal of Diabetes Research

Article Title: Autophagy Regulates Ferroptosis-Mediated Diabetic Liver Injury by Modulating the Degradation of ACSL4

doi: 10.1155/jdr/7146054

Figure Lengend Snippet: Ferroptosis inhibitor attenuated ferroptosis-related death in LO2 cells under PA treatment. (a) Representative Oil Red O staining of the LO2 cells cultured in 300 μ M PA for 24 h; scale bar: 100 μ m. (b) The quantitative analysis of Oil Red O–stained cells. (c) The viability of LO2 cells treated with PA or PA and Fer-1 was assessed. The cells were treated with PA (300 μ M) or PA (300 μ M) and Fer-1 (60 μ M) for 24 h, and then the viability of cells in each group was measured by the CCK-8 assay. (d) The level of the LDH released in LO2 cells treated with PA or PA and Fer-1 was measured using the LDH cytotoxicity assay kit. (e, f) The LO2 cells were stained with the LPO-specific dye BODIPY 581/591 C11, and LPO was detected by flow cytometry after the LO2 cells were treated with PA or PA and Fer-1. (g, h) LO2 cells were treated with PA or PA and Fer-1, stained with Annexin V-FITC and PI, and then cells that underwent apoptosis were quantified by flow cytometry; ⁣ ∗ p < 0.05, ⁣ ∗∗ p < 0.01, and ⁣ ∗∗∗ p < 0.001 between the indicated groups.

Article Snippet: Human hepatocyte LO2 cells were obtained from the China Center for Type Culture Collection (CCTCC, Wuhan, China) and cultured in Dulbecco's modified Eagle's medium (Gibco, United States) supplemented with 10% fetal bovine serum and 100 U/mL penicillin and streptomycin at 5% CO 2 .

Techniques: Staining, Cell Culture, CCK-8 Assay, LDH Cytotoxicity Assay, Flow Cytometry

Journal: Journal of Diabetes Research

Article Title: Autophagy Regulates Ferroptosis-Mediated Diabetic Liver Injury by Modulating the Degradation of ACSL4

doi: 10.1155/jdr/7146054

Figure Lengend Snippet: ACSL4 increased under PA treatment and was degraded through the autophagy-lysosomal pathway. (a) RT-qPCR analysis of the expression of ACSL4 mRNA in the LO2 cells treated with PA or PA and Fer-1. (b, c) Representative Western blots and quantitative densitometry analysis of ACSL4 protein expression in LO2 cells treated with PA or PA and Fer-1. (d, e) Protein stability of ACSL4 in LO2 cells cultured with PA, following a time course treatment with 100 μ g/mL CHX. (f, g) LO2 cells were treated with PA with or without MG132 (20 μ M) or Baf A1 (20 nM) for 8 h. Representative Western blots and quantitative densitometry analysis of the expression of the ACSL4 protein in LO2 cells. All data are presented as the mean ± Std Dev of at least three independent experiments; ⁣ ∗ p < 0.05 between the indicated groups.

Article Snippet: Human hepatocyte LO2 cells were obtained from the China Center for Type Culture Collection (CCTCC, Wuhan, China) and cultured in Dulbecco's modified Eagle's medium (Gibco, United States) supplemented with 10% fetal bovine serum and 100 U/mL penicillin and streptomycin at 5% CO 2 .

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Cell Culture

Journal: Journal of Diabetes Research

Article Title: Autophagy Regulates Ferroptosis-Mediated Diabetic Liver Injury by Modulating the Degradation of ACSL4

doi: 10.1155/jdr/7146054

Figure Lengend Snippet: Downregulation of ACSL4 reversed PA-induced ferroptosis in LO2 cells. (a) qPCR analysis of ACSL4 mRNA expression in PA300+siNC or PA300 + siACSL4 LO2 cells. (b) Immunoblotting analysis of ACSL4 protein expression in PA300+siNC or PA300+siACSL4 LO2 cells. (c) Cell viability of PA-stimulated LO2 cells transfected with control and siACSL4 knockdown RNA. (d) Rate of LPO of PA-stimulated LO2 cells transfected with control and siACSL4 knockdown RNA. Bars represent the mean ± Std Dev; ⁣ ∗∗ p < 0.01 between the indicated groups.

Article Snippet: Human hepatocyte LO2 cells were obtained from the China Center for Type Culture Collection (CCTCC, Wuhan, China) and cultured in Dulbecco's modified Eagle's medium (Gibco, United States) supplemented with 10% fetal bovine serum and 100 U/mL penicillin and streptomycin at 5% CO 2 .

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

Journal: Journal of Diabetes Research

Article Title: Autophagy Regulates Ferroptosis-Mediated Diabetic Liver Injury by Modulating the Degradation of ACSL4

doi: 10.1155/jdr/7146054

Figure Lengend Snippet: Inhibition of autophagic flux in PA-treated LO2 cells. (a, b) Representative Western blots and quantitative densitometry analysis of the expression of LC3-II, Beclin1, and p62 proteins in the LO2 cells exposed to PA for 24 h. (c, d) The LO2 cells were treated with PA for 24 h in the presence or absence of 20 nM Baf A1 during the last 2 h of coculture. Representative Western blots and quantitative densitometry analysis of the expression of the LC3-II protein in LO2 cells. (e) Representative images of LO2 cells infected with mRFP-GFP-LC3 and exposed to PA; Green: GFP puncta; Red: mRFP puncta. (f) GFP dots and mRFP dots were counted in each cell. (g) Semiquantitative analysis of autophagosomes (yellow puncta in merged images) and autolysosomes (red puncta in merged images). All data are presented as the mean ± Std Dev of at least three separate experiments; ns: nonsignificant; ⁣ ∗ p < 0.05, ⁣ ∗∗ p < 0.01, and ⁣ ∗∗∗ p < 0.001 between the indicated groups.

Article Snippet: Human hepatocyte LO2 cells were obtained from the China Center for Type Culture Collection (CCTCC, Wuhan, China) and cultured in Dulbecco's modified Eagle's medium (Gibco, United States) supplemented with 10% fetal bovine serum and 100 U/mL penicillin and streptomycin at 5% CO 2 .

Techniques: Inhibition, Western Blot, Expressing, Infection

Journal: Journal of Diabetes Research

Article Title: Autophagy Regulates Ferroptosis-Mediated Diabetic Liver Injury by Modulating the Degradation of ACSL4

doi: 10.1155/jdr/7146054

Figure Lengend Snippet: Autophagy may be involved in ferroptosis by regulating the expression of ACSL4. (a) CCK-8 assay was performed to determine the viability of LO2 cells treated with various concentrations of 3-MA for 24 h. (b) The level of the LDH released in LO2 cells treated with various concentrations of 3-MA for 24 h was measured using the LDH cytotoxicity assay kit. (c, d) LO2 cells were treated with 5 mM 3-MA for 24 h and stained with the LPO-specific dye BODIPY 581/591 C11, and LPO was detected by flow cytometry. (e, f) LO2 cells were treated with 5 mM 3-MA for 24 h and stained with Annexin V-FITC and PI, and then the cells that underwent apoptosis were quantified by flow cytometry. (g, h) Representative Western blots and quantitative densitometry analysis of the expression of the ACSL4 and LC3-II proteins in LO2 cells cultured with 3-MA for 24 h. (i, j) LO2 cells were treated with BSA, BSA+Rap (50 nM), PA (300 μ M), or PA (300 μ M)+Rap (50 nM) for 24 h. Representative Western blots and quantitative densitometry analysis of the expression of ACSL4 and LC3 proteins in LO2 cells. All data are presented as the mean ± Std Dev of at least three separate experiments; ns: nonsignificant; ⁣ ∗ p < 0.05 and ⁣ ∗∗ p < 0.01 between the indicated groups.

Article Snippet: Human hepatocyte LO2 cells were obtained from the China Center for Type Culture Collection (CCTCC, Wuhan, China) and cultured in Dulbecco's modified Eagle's medium (Gibco, United States) supplemented with 10% fetal bovine serum and 100 U/mL penicillin and streptomycin at 5% CO 2 .

Techniques: Expressing, CCK-8 Assay, LDH Cytotoxicity Assay, Staining, Flow Cytometry, Western Blot, Cell Culture

Journal: Bioengineered

Article Title: Long non-coding RNA AC012668 suppresses non-alcoholic fatty liver disease by competing for microRNA miR-380-5p with lipoprotein-related protein LRP2

doi: 10.1080/21655979.2021.1960463

Figure Lengend Snippet: NAFLD models established to investigate changes in AC012668 expression. (a) Heat map of differentially expressed lncRNAs in NAFLD. (b) Cell viability and (c and d) apoptosis of LO2 cells treated with FFA. (e and f) Oil-Red O staining image and TG level in the liver tissues of HFD mice. (g) AC012668 expression levels in the liver tissues of HFD mice. (h) Expression of AC012668 in FFA-treated cells. *P < 0.05 vs. control; **P < 0.01 vs. control

Article Snippet: Normal human hepatocyte lines LO2 were purchased from Biowit Biotechnology Inc. (cat no. C0009).

Techniques: Expressing, Staining

Journal: Bioengineered

Article Title: Long non-coding RNA AC012668 suppresses non-alcoholic fatty liver disease by competing for microRNA miR-380-5p with lipoprotein-related protein LRP2

doi: 10.1080/21655979.2021.1960463

Figure Lengend Snippet: AC012668 suppresses TG/lipid accumulation and lipogenesis in LO2 cells. (a) AC012668 expression level in FFA-treated LO2 cells transfected with the AC012668 overexpression plasmid. (b) mRNA and (c and d) protein expression levels of SCD1, SREBP1 , and FAS in FFA-treated LO2 cells. (e) TG level and (f) Oil-Red O staining in FFA-treated LO2 cells. *P < 0.05 vs. pcDNA3.1 or AC012668.

Article Snippet: Normal human hepatocyte lines LO2 were purchased from Biowit Biotechnology Inc. (cat no. C0009).

Techniques: Expressing, Transfection, Over Expression, Plasmid Preparation, Staining

Journal: Bioengineered

Article Title: Long non-coding RNA AC012668 suppresses non-alcoholic fatty liver disease by competing for microRNA miR-380-5p with lipoprotein-related protein LRP2

doi: 10.1080/21655979.2021.1960463

Figure Lengend Snippet: miR-380-5p directly targets AC012668 . (a) Wild and mutant types of AC012668 reporters labeled with luciferase. (b) Expression levels of miR-380-5p in AC012668 -enhanced/-inhibited LO2 cells. (c) Relative luciferase activities in wild-type and mutant AC012668 groups compared with the mimic negative control group. (d) Relative enrichment of AC012668 in the biotinylated miR-380-5p group. (e) AC012668 expression levels in the liver tissues of HFD mice. (f) Expression level of miR-380-5p in LO2 cells treated with 1 mM FFA. *P < 0.05 vs. biotin-NC; **P < 0.01 vs. si-NC, vector, mimic NC, or control

Article Snippet: Normal human hepatocyte lines LO2 were purchased from Biowit Biotechnology Inc. (cat no. C0009).

Techniques: Mutagenesis, Labeling, Luciferase, Expressing, Negative Control, Plasmid Preparation

Journal: Bioengineered

Article Title: Long non-coding RNA AC012668 suppresses non-alcoholic fatty liver disease by competing for microRNA miR-380-5p with lipoprotein-related protein LRP2

doi: 10.1080/21655979.2021.1960463

Figure Lengend Snippet: miR-380-5p promotes TG/lipid accumulation and lipogenesis in LO2 cells transfected with AC012668 . (a) AC012668 expression detected using reverse transcription-quantitative polymerase chain reaction. (b) mRNA and (c and d) protein levels of SCD1, SREBP1 , and FAS . (e) TG level and (f) lipid deposition. *P < 0.05 vs. AC012668 + mimic NC; **P < 0.01 vs. vector

Article Snippet: Normal human hepatocyte lines LO2 were purchased from Biowit Biotechnology Inc. (cat no. C0009).

Techniques: Transfection, Expressing, Real-time Polymerase Chain Reaction, Plasmid Preparation

Journal: Bioengineered

Article Title: Long non-coding RNA AC012668 suppresses non-alcoholic fatty liver disease by competing for microRNA miR-380-5p with lipoprotein-related protein LRP2

doi: 10.1080/21655979.2021.1960463

Figure Lengend Snippet: LRP2 is a target of miR-380-5p . (a) Wild and mutant types of LRP2 reporters labeled with luciferase. (b) mRNA expression of LRP2 in FFA-treated LO2 cells transfected with the miR-380-5p mimic or inhibitor. (c) Relative luciferase activities in wild-type and mutant LRP2 groups compared with the mimic negative control group. (d) Relative enrichment of LRP2 in the biotinylated miR-380-5p group. (e) AC012668 expression levels in the liver tissues of HFD mice. (f) Expression level of LRP2 in FFA-treated LO2 cells. *P < 0.05 vs. biotin-NC; **P < 0.01 vs. inhibitor NC, mimic NC, or control

Article Snippet: Normal human hepatocyte lines LO2 were purchased from Biowit Biotechnology Inc. (cat no. C0009).

Techniques: Mutagenesis, Labeling, Luciferase, Expressing, Transfection, Negative Control

Journal: Bioengineered

Article Title: Long non-coding RNA AC012668 suppresses non-alcoholic fatty liver disease by competing for microRNA miR-380-5p with lipoprotein-related protein LRP2

doi: 10.1080/21655979.2021.1960463

Figure Lengend Snippet: Effects of LRP2 knockdown on TG/lipid accumulation and lipogenesis in miR-380-5p -inhibited LO2 cells. (a) miR-380-5p expression level in LO2 cells cotransfected with the miR-380-5p inhibitor and si-LRP2 . (b) mRNA and (c and d) protein levels of SCD1, SREBP1 , and FAS in the miR-380-5p inhibitor- and si-LRP2 -cotransfected cells. (e) TG level and (f) lipid deposition in the miR-380-5p inhibitor- and si-LRP2 -cotransfected cells. *P < 0.05 vs. control or miR-380-5p mimic + si-NC

Article Snippet: Normal human hepatocyte lines LO2 were purchased from Biowit Biotechnology Inc. (cat no. C0009).

Techniques: Expressing

Journal: Molecules

Article Title: Isolation, Characterization, Moisturization and Anti-HepG2 Cell Activities of a Novel Polysaccharide from Cyanobacterium aponinum

doi: 10.3390/molecules29194556

Figure Lengend Snippet: Cytotoxicity activity of CAP on LO2 and HepG2 cells in vitro. The cell viability of LO2 treated with different concentrations of CAP (0.0, 0.1, 0.5, 1.0, 5.0, 10.0, 20.0, 50.0, 100.0 µg mL −1 ) for 48 h ( a ) and the growth inhibition rate of CAP with same concentrations on HepG2 cell after 48 h treatment. Gemcitabine was used as a positive control ( b ).

Article Snippet: The cytotoxicity of CAP on human fetal hepatocyte cell line LO2 (purchased from iCell Bioscience Inc., Shanghai, China) was evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.

Techniques: Activity Assay, In Vitro, Inhibition, Positive Control