Journal: Cancer Communications

Article Title: Intranuclear paraspeckle‐circular RNA TACC3 assembly forms RNA‐DNA hybrids to facilitate MASH‐related hepatocellular carcinoma growth in an m 6 A‐dependent manner

doi: 10.1002/cac2.70061

Figure Lengend Snippet: m 6 A‐modified circTACC3 is up‐regulated in MASH‐related HCC. (A) Schematic representation of m 6 A‐circRNA epitranscriptomic microarray assay. (B) Hierarchical clustering heatmap of differentially m 6 A modified circRNAs from MASH‐related HCC tumor and paired peritumoral normal tissues (m 6 A‐circRNA epitranscriptomic microarray assay; absolute m 6 A modification quantity shown, n = 5). (C) Venn diagram of overlapping circRNAs in MASH‐related HCC tumors with concurrent increases in absolute m 6 A modification quantity, relative m 6 A modification rate, and relative expression levels. (D‐E) The circTACC3 level in MASLD tissues, MASH‐related HCC tumor tissues, and paired peritumoral normal tissues ( n = 62) determined by ISH assay (D) and corresponding expression score analysis (E). ** P < 0.01; *** P < 0.001; NS , not significant. (F) MeRIP assay shows the enrichment of m 6 A‐modified circTACC3 in MASLD tissues, MASH‐related HCC tumor tissues and paired peritumoral normal tissues ( n = 3). (G) 3D‐FISH performed on MASH‐related HCC tumor and paired peritumoral normal tissue derived organoids. Left upper panel shows circTACC3 (red) and DAPI (blue). Left lower panel shows the merge in 3D view. Right upper panel shows representative z‐stack layer capture. Right lower panel shows depth coding. (H) Nuclear‐cytoplasmic fractionation assay determined circTACC3 expression in nuclear and cytoplasmic fractionation, respectively ( n = 4). ** P < 0.01; *** P < 0.001; NS , not significant. (I) Schematic representation of exon 4 back‐splicing, circTACC3 forming, and the design of indicated primers. (J) Electrophoresis of RT‐PCR product amplified from cDNA or gDNA. (K) The expression of circTACC3 and TACC3 homologous mRNA from RNA with or without RNase R treatment. (L) Levels of circTACC3 and TACC3 mRNA in indicated cells that were treated with or without actinomycin D ( n = 4). *** P < 0.001. Abbreviations: m 6 A, N6‐methyladenosine; MASLD, metabolic dysfunction‐associated steatotic liver disease; MASH, metabolic dysfunction‐associated steatohepatitis; HCC, hepatocellular carcinoma; T, tumor tissues; PT, peritumoral normal tissue; ISH, in situ hybridization‐ immunofluorescence; MeRIP, methylated RNA immunoprecipitation; 3D, three dimensions; FISH, fluorescence in situ hybridization; NAS, non‐alcoholic fatty liver disease activity score; RT‐PCR, reverse transcription‐PCR.

Article Snippet: To generate stable cell lines with the inducible overexpression of RNase H1, HepG2 and HCCLM3 cells were infected with lentivirus expressing TetIIP‐RNase H1‐3FLG‐Ubi‐TetR‐IRES‐Puromysin and selected with puromycin (# T19978 , TargetMol, MA, US, final concentration 2 μg/mL) for 1‐2 weeks.

Techniques: Modification, Microarray, Expressing, In Situ Hybridization, Derivative Assay, Fractionation, Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Amplification, Immunofluorescence, Methylation, RNA Immunoprecipitation, Fluorescence, Activity Assay, Reverse Transcription

Journal: Cancer Communications

Article Title: Intranuclear paraspeckle‐circular RNA TACC3 assembly forms RNA‐DNA hybrids to facilitate MASH‐related hepatocellular carcinoma growth in an m 6 A‐dependent manner

doi: 10.1002/cac2.70061

Figure Lengend Snippet: circTACC3‐R loop formation is regulated by lipid overload and m 6 A modification. (A) 3D‐distribution of circTACC3 (glow) and S9.6‐stained R loops (spectrum) in nuclei (blue) of MASH‐HCC tissue derived organoids after indicated treatment. (B‐C) DRIP assay shows the enrichment of circTACC3 in R loop structure following m 6 A modification interference in PA and OA treated HCCLM3 and HepG2 cells ( n = 3). * P < 0.05; ** P < 0.01. (D) Representative fluorescence images show S9.6‐stained R loops (green) in PA + OA induced HCC cells transfected with the RNase H1‐Tet‐On system after treatment with or without Dox. (E) Nucleo‐plasmic fractionation shows the altered intracellular localization of circTACC3 in PA and OA treated HCCLM3 and HepG2 cells ( n = 4). *** P < 0.001. (F‐G) Representative fluorescence pictures (F) and peak graphs of the linear ROI (G) demonstrating the colocalization of circTACC3 (red), S9.6‐indicated R loops (green), and NONO/p54 nrb (yellow) in nuclei (blue) with or without Dox‐inducible RNase H1 expression in PA and OA treated HCCLM3 and HepG2 cells. Abbreviations: MASH, metabolic dysfunction‐associated steatohepatitis; HCC, hepatocellular carcinoma; PA, palmitic acid; OA, oleic acid; T, tumor tissue; PT, peritumoral normal tissue; SAH, S‐adenosylhomocysteine; DAA, 3‐deazaadenosine; DRIP, DNA‐RNA immunoprecipitation; Dox, Doxycycline; ROI, region of interest.

Article Snippet: To generate stable cell lines with the inducible overexpression of RNase H1, HepG2 and HCCLM3 cells were infected with lentivirus expressing TetIIP‐RNase H1‐3FLG‐Ubi‐TetR‐IRES‐Puromysin and selected with puromycin (# T19978 , TargetMol, MA, US, final concentration 2 μg/mL) for 1‐2 weeks.

Techniques: Modification, Staining, Derivative Assay, Fluorescence, Transfection, Fractionation, Expressing, RNA Immunoprecipitation

Journal: Cancer Communications

Article Title: Intranuclear paraspeckle‐circular RNA TACC3 assembly forms RNA‐DNA hybrids to facilitate MASH‐related hepatocellular carcinoma growth in an m 6 A‐dependent manner

doi: 10.1002/cac2.70061

Figure Lengend Snippet: DSB‐circTACC3‐R loops aggregated to promote the inter‐TADs contact. (A) After DRIP‐ChIRP sequencing, the reads distributions across peaks of all independent biological replicates are presented. (B) Genome‐wide distribution of the circTACC3‐R Loop‐located genes positively correlated with PA + OA induction or negatively correlated with m 6 A modification intervention. (C) Representative fluorescence images of the colocalization of indicated molecules in nuclei of HCC cells treated with or without PA and OA following RNase R treatment. (D) Schematic representation shows combination of DRIP‐ChIRP‐seq and γH2AX CUT&Tag‐seq to analyze the distribution of the DSB‐circTACC3‐R Loop structures in the genome. (E) Genome‐wide distribution of the DSB‐circTACC3‐R loop located genes in PA + OA induced HepG2 cells. (F) Top four enriched DSB‐circTACC3‐R loop‐binding motifs based on de novo motif analysis. (G) The dynamic clustering of paraspeckles (indicated by NONO/p54 nrb ‐mCherry) were filmed using STELLARIS Dynamic Signal Enhancement 24 h after PA + OA induction at 5‐min intervals for a duration of 1.5 h. Examples (from 50 min to 85 min) of fusions of several NONO/p54 nrb ‐mCherry foci are shown (time points indicated in minutes). (H) Heatmap depicting the fold change(log 2 ) in Hi‐C contact frequencies between PA + OA‐treated and control cells throughout chromosome 7. Interactions that increase in PA + OA group (red) or decrease in Mock group (blue) are evident. Profile of DRIP‐ChIRP‐seq and γH2AX CUT&Tag‐seq are shown on the top. TADs that had higher inter‐TADs contact frequencies (named “contact‐elevated TADs”) in both long‐range (green box) and between adjacent TADs (red box) are marked. DSB‐circTACC3‐R loops are marked with red arrow. (I) Hi‐C maps around the human STX6 locus that formed DSB‐circTACC3‐R loop structure are shown. DSB‐circTACC3‐R loops are marked with red arrow. Abbreviations: DRIP, DNA‐RNA immunoprecipitation; ChIRP, chromatin isolation by RNA purification; γH2AX, Ser‐139 residue of the histone variant H2AX; CUT&Tag, cleavage under targets and tagmentation; IF, immunofluorescence; FISH, fluorescence i n situ hybridization; PA, palmitic acid; OA, oleic acid; Hi‐C, high‐throughput/resolution chromosome conformation capture; DSB, DNA double‐strand breaks; STX6, Syntaxin 6.

Article Snippet: To generate stable cell lines with the inducible overexpression of RNase H1, HepG2 and HCCLM3 cells were infected with lentivirus expressing TetIIP‐RNase H1‐3FLG‐Ubi‐TetR‐IRES‐Puromysin and selected with puromycin (# T19978 , TargetMol, MA, US, final concentration 2 μg/mL) for 1‐2 weeks.

Techniques: Sequencing, Genome Wide, Modification, Fluorescence, Binding Assay, Hi-C, Control, RNA Immunoprecipitation, Isolation, Purification, Residue, Variant Assay, Immunofluorescence, Hybridization, High Throughput Screening Assay

Journal: Cancer Communications

Article Title: Intranuclear paraspeckle‐circular RNA TACC3 assembly forms RNA‐DNA hybrids to facilitate MASH‐related hepatocellular carcinoma growth in an m 6 A‐dependent manner

doi: 10.1002/cac2.70061

Figure Lengend Snippet: DSB‐circTACC3‐R loop‐localized genes are selectively activated. (A) List of DSB‐circTACC3‐R loop‐localized genes. (B) DSB‐circTACC3‐loop‐localized genes expression in HepG2 cells with/without lipid overload induction ( n = 4). * P < 0.05; ** P < 0.01; *** P < 0.001; NS , not significant. (C‐D) DRIP‐ChIRP‐seq (C) and γH2AX CUT&Tag‐seq (D) RPKM analysis of DSB‐circTACC3‐loop‐localized genes to compare circTACC3‐R Loop enrichment within the “contact‐elevated TADs” ( n = 26) or not within the “contact‐elevated TADs” ( n = 12). * P < 0.05; ** P < 0.01; *** P < 0.001; NS , not significant. (E‐F) Representative fluorescence images (E) and peaks graphs of the linear ROI (F) show the colocalization of indicated molecules with or without Dox‐inducible RNase H1 expression. (G) NONO/p54 nrb ‐mCherry HepG2 cells with or without Dox‐inducible RNase H1 expression were filmed 24 h after PA + OA induction at 5‐min intervals. Abbreviations: PA, palmitic acid; OA, oleic acid; TAD, topologically associated domain; γH2AX, Ser‐139 residue of the histone variant H2AX; CUT&Tag, cleavage under targets and tagmentation; RPKM, reads per kilobase per million mapped reads; Dox, Doxycycline; ROI, region of interest.

Article Snippet: To generate stable cell lines with the inducible overexpression of RNase H1, HepG2 and HCCLM3 cells were infected with lentivirus expressing TetIIP‐RNase H1‐3FLG‐Ubi‐TetR‐IRES‐Puromysin and selected with puromycin (# T19978 , TargetMol, MA, US, final concentration 2 μg/mL) for 1‐2 weeks.

Techniques: Expressing, Fluorescence, Residue, Variant Assay

Journal: Cell Biology and Toxicology

Article Title: Gestational bisphenol A exposure induces fatty liver development in male offspring mice through the inhibition of HNF1b and upregulation of PPARγ

doi: 10.1007/s10565-020-09535-3

Figure Lengend Snippet: Effects of gestational BPA exposure on the expression of key regulators involved in in vivo lipid metabolism. Genes involved in lipid metabolism regulation in the liver were determined in male offspring. a These genes include Srebp1 , FASN , ACC-1 , SCD-1 , and PPARα ; ** p < 0.01, indicate significant differences when compared with respective controls. The mRNA and protein expression levels of PPARγ and HNF1b were determined in the liver tissues of b – f male and g – k female offspring using qRT-PCR and immunoblotting. * p < 0.05; ** p < 0.01, indicate significant differences when compared between the two groups

Article Snippet: After separation of offspring mice, different genders were randomly divided into five equal groups including a control group, high fat diet (HFD) group, BPA + HFD group, BPA + Rh1 (ginsenoside Rh1, inhibitor of PPARγ, MedChemExpress) group, and BPA group.

Techniques: Expressing, In Vivo, Quantitative RT-PCR, Western Blot

Journal: Cell Biology and Toxicology

Article Title: Gestational bisphenol A exposure induces fatty liver development in male offspring mice through the inhibition of HNF1b and upregulation of PPARγ

doi: 10.1007/s10565-020-09535-3

Figure Lengend Snippet: Upregulation of PPARγ is involved in gestational BPA exposure-induced effects in male offspring. Determination of a liver TG content and h H&E staining of liver tissues are shown. Frozen sections were stained using ( c Oil Red O and d BODIPY to observe lipid droplets. Size bar, 50 μm. Both mRNA and protein expression levels of PPARγ and HNF1b were determined in the liver tissues of ( e – i ) male and ( j – n ) female mice using qRT-PCR and immunoblotting. * p < 0.05; ** p < 0.01, indicate significant differences when compared between the two groups

Article Snippet: After separation of offspring mice, different genders were randomly divided into five equal groups including a control group, high fat diet (HFD) group, BPA + HFD group, BPA + Rh1 (ginsenoside Rh1, inhibitor of PPARγ, MedChemExpress) group, and BPA group.

Techniques: Staining, Expressing, Quantitative RT-PCR, Western Blot

Journal: Cell Biology and Toxicology

Article Title: Gestational bisphenol A exposure induces fatty liver development in male offspring mice through the inhibition of HNF1b and upregulation of PPARγ

doi: 10.1007/s10565-020-09535-3

Figure Lengend Snippet: Both estrogen and PPARγ regulate BPA-induced glucose and lipid metabolism in vitro. a – c Both mRNA expression and protein expression of PPARγ were determined in LV-shPPARγ. ** p < 0.01, indicate significant differences when compared with respective controls. d Fatty acid uptake was analyzed as well as e Oil Red O staining and f BODIPY staining were both performed to observe lipid accumulation in L02 cells. g Glycogen content and h glucose uptake were determined to evaluate dysfunction in glucose metabolism. i , j The mRNA expression levels of PPARγ and HNF1b were determined in L02 cells using qRT-PCR and immunoblotting. * p < 0.05; ** p < 0.01, indicate significant differences when compared between the two groups

Article Snippet: After separation of offspring mice, different genders were randomly divided into five equal groups including a control group, high fat diet (HFD) group, BPA + HFD group, BPA + Rh1 (ginsenoside Rh1, inhibitor of PPARγ, MedChemExpress) group, and BPA group.

Techniques: In Vitro, Expressing, Staining, Quantitative RT-PCR, Western Blot

Journal: Cell Biology and Toxicology

Article Title: Gestational bisphenol A exposure induces fatty liver development in male offspring mice through the inhibition of HNF1b and upregulation of PPARγ

doi: 10.1007/s10565-020-09535-3

Figure Lengend Snippet: The downregulation of HNF1b is involved in BPA-induced glucose/lipid metabolic dysfunction. a , b HNF1b binding on the promoters of PPARγ in L02 cells was detected using ChIP, ** p < 0.01 indicates significant differences when compared with control, and ## p < 0.01 indicates significant differences when compared with BPA. c Fatty acid uptake was analyzed as well as d Oil Rred O staining and e BODIPY staining were performed to observe lipid accumulation in L02 cells. f Glycogen content and g glucose uptake were both analyzed in LV-transfected L02 cells to evaluate dysfunction in glucose metabolism. h – l Both mRNA and protein expression levels of PPARγ and HNF1b were determined using qRT-PCR and immunoblotting. * p < 0.05; ** p < 0.01, indicate significant differences when compared between the two groups

Article Snippet: After separation of offspring mice, different genders were randomly divided into five equal groups including a control group, high fat diet (HFD) group, BPA + HFD group, BPA + Rh1 (ginsenoside Rh1, inhibitor of PPARγ, MedChemExpress) group, and BPA group.

Techniques: Binding Assay, Control, Staining, Transfection, Expressing, Quantitative RT-PCR, Western Blot

Journal: Journal of Ginseng Research

Article Title: Effects of Natural Bioactive Products on the Growth and Ginsenoside Contents of Panax ginseng Cultured in an Aeroponic System

doi: 10.5142/jgr.2012.36.4.430

Figure Lengend Snippet: Protopanaxadiol (PD)/ protopanaxatriol (PT) ratio of Panax ginseng cultured with the aeroponic system using a two-layer vertical type of nutrient bath. P. ginseng was cultured by the aeroponic system in the lower and the upper layers in a two-layer type of nutrient bath. PD/PT means the ratio of PD ginsenosides and PT ginsenosides. PD: Rb1, Rb2, Rc, Rd; PT: Re, Rf, Rg2, Rh1. T1-T5, natural bioactive products. T1, Manda enzyme (×10,000); T2, Yangmyeongwon (×8,000); T3, effective microorganisms (×3,000); T4, Kelpak (×3,000); T5, control. The mean values±SD obtained from triplicate separate experiments are shown.

Article Snippet: Ginsenoside components such as ginsenoside Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, Rg2, and Rh1 were purchased from a chemical company (ChromaDex Inc., Santa Anna, CA, USA).

Techniques: Cell Culture

Journal: Nutrients

Article Title: Ginseng Saponin Enriched in Rh1 and Rg2 Ameliorates Nonalcoholic Fatty Liver Disease by Inhibiting Inflammasome Activation

doi: 10.3390/nu13030856

Figure Lengend Snippet: Saponin extract has high contents of ginsenoside Rh1 and Rg2 that exert an anti-inflammatory effect. The mRNA expression of inflammatory cytokines ( A ) Arg1, ( B ) Ccl2, ( C ) Ccl4, and ( D ) Cxcl2 were measured by qRT-PCR in the liver tissue. ( E ) The protein level of TNF-α in liver tissue lysates were determined by ELISA. ( F ) The level of ALT in the serum was measured. The mRNA expression of inflammatory cytokines in ImKCs, compared with saponin extract and red ginseng, ( G) Arg1, ( H ) Ccl2, and ( I ) IL-1β. ( J ) The fold changes of different compounds in saponin extract and red ginseng. The mRNA expression of inflammatory cytokines in ImKCs, compared with ginsenoside Rh1 and Rg2, ( K ) TNF-α, ( L ) Ccl2, ( M ) Arg1, and ( N ) IL-10. Relative mRNA expression levels were normalized to mouse GAPDH levels. The data are expressed as means ± sem. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Dulbecco’s Modified Eagle’s Medium (DMEM), Corning ® , Glendale, CA, USA; Fetal Bovine Serum (FBS), Corning ® , Glendale, USA; M199 medium, Corning ® , CA, USA; Penicillin-Streptomycin solution 100× (PS), Corning ® , CA, USA; TB Green Premix Ex Taq II, TAKARA, Shiga, Japan; Lipopolysaccharide (LPS), Sigma-Aldrich, St. Louis, MO, USA; Carbonyl Cyanide m-Chlorophenylhydrazone (CCCP), Sigma-Aldrich, St. Louis, USA; Palmitic Acid, Sigma-Aldrich, St. Louis, USA; Oil red O, Sigma-Aldrich, St. Louis, USA; Pierce™ BCA Protein Assay Kit, Thermo Scientific, Waltham, MA, USA; Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IkBα), Santa Cruz, CA, USA; Phosphorylated-IkBα, Santa Cruz, CA, USA; Heat shock protein 60, Santa Cruz, CA, USA; Translocase of outer mitochondrial membrane 40 homolog, Santa Cruz, CA, USA; Mitochondrial import inner membrane translocase, Santa Cruz, CA, USA; Actin antibodies, Santa Cruz, CA, USA; Primer Script TM RT Reagent Kit with gDNA Eraser, Shiga, Japan; ATP, Sigma-Aldrich, St. Louis, USA; Metformin, Sigma-Aldrich, St. Louis, USA; NLRP3 inhibitor, Selleckchem, Houston, TX, USA; Ginsenoside Rh1, Merck company, Kenilworth, NJ, USA and Ginsenoside Rg2, Merck company, NJ, USA;

Techniques: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Nutrients

Article Title: Ginseng Saponin Enriched in Rh1 and Rg2 Ameliorates Nonalcoholic Fatty Liver Disease by Inhibiting Inflammasome Activation

doi: 10.3390/nu13030856

Figure Lengend Snippet: Ginsenoside Rh1 and Rg2 treatment inhibits the activation of the NLRP3 inflammasome. ( A ) Representative Western blotting analysis of IkB, p-IkB, and Actin. Saponin extract 62.5 µg/mL is referred to as SapH and Saponin extract 31.25 µg/mL as SapL. The mRNA expression of IL-1β in ( B ) liver tissue and ( C ) in ImKCs. ( D ) The protein level of IL-1β in liver tissue lysates was determined by ELISA. The mRNA expression of the NLRP3 inflammasome ( E ) and Aim2 inflammasome ( F ) in liver tissue and in ImKCs ( G , H ). The mRNA expression of IL-1β ( I ) in ImKCs. The mRNA expression of NLRP3 inflammasome treated with ginsenoside Rh1 ( J ) and Rg2 ( K) in ImKCs. The mRNA expression of IL-1β treated with ginsenoside Rh1 ( L ) and Rg2 ( M ) in ImKCs. The protein level of IL-1β treated with ginsenoside Rh1 ( N ) and Rg2 ( O ) in the cell culture was determined by ELISA. Relative mRNA expression levels were normalized to mouse GAPDH levels. The data are expressed as means ± sem. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Dulbecco’s Modified Eagle’s Medium (DMEM), Corning ® , Glendale, CA, USA; Fetal Bovine Serum (FBS), Corning ® , Glendale, USA; M199 medium, Corning ® , CA, USA; Penicillin-Streptomycin solution 100× (PS), Corning ® , CA, USA; TB Green Premix Ex Taq II, TAKARA, Shiga, Japan; Lipopolysaccharide (LPS), Sigma-Aldrich, St. Louis, MO, USA; Carbonyl Cyanide m-Chlorophenylhydrazone (CCCP), Sigma-Aldrich, St. Louis, USA; Palmitic Acid, Sigma-Aldrich, St. Louis, USA; Oil red O, Sigma-Aldrich, St. Louis, USA; Pierce™ BCA Protein Assay Kit, Thermo Scientific, Waltham, MA, USA; Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IkBα), Santa Cruz, CA, USA; Phosphorylated-IkBα, Santa Cruz, CA, USA; Heat shock protein 60, Santa Cruz, CA, USA; Translocase of outer mitochondrial membrane 40 homolog, Santa Cruz, CA, USA; Mitochondrial import inner membrane translocase, Santa Cruz, CA, USA; Actin antibodies, Santa Cruz, CA, USA; Primer Script TM RT Reagent Kit with gDNA Eraser, Shiga, Japan; ATP, Sigma-Aldrich, St. Louis, USA; Metformin, Sigma-Aldrich, St. Louis, USA; NLRP3 inhibitor, Selleckchem, Houston, TX, USA; Ginsenoside Rh1, Merck company, Kenilworth, NJ, USA and Ginsenoside Rg2, Merck company, NJ, USA;

Techniques: Activation Assay, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Nutrients

Article Title: Ginseng Saponin Enriched in Rh1 and Rg2 Ameliorates Nonalcoholic Fatty Liver Disease by Inhibiting Inflammasome Activation

doi: 10.3390/nu13030856

Figure Lengend Snippet: Ginsenoside Rh1 and Rg2 alleviated the activation of NLRP3 by promoting mitophagy. ( A ) ImKCs were measured for MitoSOX 3 h after treatment with LPS (500 ng/mL) and ginsenoside Rh1 (62.5 µg/mL) and Rg2 (62.5 µg/mL). ( B ) Hep3B cells were transfected with pLVX-puro-mt-Keima. Analysis of expressing mt-Keima treated with ginsenoside Rh1 and Rg2 for 24 h. Rh1 and Rg2 mediated mitophagy was quantified by flow cytometry with 2-µM carbonyl cyanide m-chlorophenylhydrazone (CCCP); the excitation wavelength was 586 nm in a neutral and 440 nm in an acidic environment. ( C ) Representative Western blotting analysis of Tim23, Tom40, and HSP60. The concentration of ginsenoside Rh1 and Rg2 was 62.5 µg/mL. ( D ) Ginsenoside Rh1 and Rg2-mediated mitophagy was quantified by flow cytometry with 62.5-µM metformin for 24 h in mt-Keima Hep3B cells. The mRNA expression of IL-1β in ImKCs cotreated with LPS+ATP, Metformin, and Rh1 ( E ) and Rg2 ( F ). The mRNA expression of the NLRP3 inflammasome in ImKCs cotreated with LPS+ATP, Metformin, and Rh1 ( G ) and Rg2 ( H ). Relative mRNA expression levels were normalized to mouse GAPDH levels. The data are expressed as means ± sem. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Dulbecco’s Modified Eagle’s Medium (DMEM), Corning ® , Glendale, CA, USA; Fetal Bovine Serum (FBS), Corning ® , Glendale, USA; M199 medium, Corning ® , CA, USA; Penicillin-Streptomycin solution 100× (PS), Corning ® , CA, USA; TB Green Premix Ex Taq II, TAKARA, Shiga, Japan; Lipopolysaccharide (LPS), Sigma-Aldrich, St. Louis, MO, USA; Carbonyl Cyanide m-Chlorophenylhydrazone (CCCP), Sigma-Aldrich, St. Louis, USA; Palmitic Acid, Sigma-Aldrich, St. Louis, USA; Oil red O, Sigma-Aldrich, St. Louis, USA; Pierce™ BCA Protein Assay Kit, Thermo Scientific, Waltham, MA, USA; Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IkBα), Santa Cruz, CA, USA; Phosphorylated-IkBα, Santa Cruz, CA, USA; Heat shock protein 60, Santa Cruz, CA, USA; Translocase of outer mitochondrial membrane 40 homolog, Santa Cruz, CA, USA; Mitochondrial import inner membrane translocase, Santa Cruz, CA, USA; Actin antibodies, Santa Cruz, CA, USA; Primer Script TM RT Reagent Kit with gDNA Eraser, Shiga, Japan; ATP, Sigma-Aldrich, St. Louis, USA; Metformin, Sigma-Aldrich, St. Louis, USA; NLRP3 inhibitor, Selleckchem, Houston, TX, USA; Ginsenoside Rh1, Merck company, Kenilworth, NJ, USA and Ginsenoside Rg2, Merck company, NJ, USA;

Techniques: Activation Assay, Transfection, Expressing, Flow Cytometry, Western Blot, Concentration Assay