Journal: International Journal of Molecular Sciences

Article Title: Novel Benzoxazoles Containing 4-Amino-Butanamide Moiety Inhibited LPS-Induced Inflammation by Modulating IL-6 or IL-1β mRNA Expression

doi: 10.3390/ijms23105331

Figure Lengend Snippet: Compounds 5f and 4d attenuate inflammation in vitro. Effects of compounds (10 µM) on the protein levels of STAT3, IκB, and NF-κB in AML-12 cells. ( a ) Expression levels of inflammation related-proteins in vitro. ( b – d ) Protein expression levels were normal-ized against the indicated protein. * p < 0.05 compared with the group treated with the PBS (vehicle). + p < 0.05 compared with the group treated with the LPS. Data are pre-sented as the mean ± SD.

Article Snippet: The human keratinocytes HaCaT or the alpha mouse liver 12 (AML-12) cells were obtained from Cell Lines Service GmbH (Eppelheim, Germany).

Techniques: In Vitro, Expressing

Journal: Cells

Article Title: Ursodeoxycholic Acid Regulates Hepatic Energy Homeostasis and White Adipose Tissue Macrophages Polarization in Leptin-Deficiency Obese Mice

doi: 10.3390/cells8030253

Figure Lengend Snippet: Ursodeoxycholic acid (UDCA) alleviates high free fatty acid (HFFA)-induced hepatocyte lipogenesis, reactive oxygen species (ROS) production, and mitochondrial dysfunction in AML12 cells. AML12 cells were treated with 1 mM HFFA with 10, 30, 100 μM UDCA. ( A ) Lipid accumulation display using Oil Red O stain (red). ROS levels were measured using DCFH-DA (green) stain. Images of AML12 cells stained with Mito Tracker for mitochondria (red). qRT-PCR analysis of ( B ) Complex I, II, III, IV, and V mRNA expression in AML12 cells. Relative mRNA expression was normalized to Gapdh and then normalized to the controls. ( C ) Immunofluorescence analysis of SREBP1c (green), CD36 (red), NF-κB (green), and FXR (green) expression, and DAPI (blue) for nuclear. Scale bar, 25 μm. qRT-PCR analysis of ( D ) Srebp-1c, Fas , and Scd-1 mRNA expression in AML12 cells. In all panels, results are expressed as the mean ± S.E.M. of five independent experiments, and statistical significance of differences between means was assessed using an unpaired Student’s t -test (* p ≤ 0.05; 0 mM HFFA vs. 1 mM HFFA. # p ≤ 0.05; 1 mM HFFA vs. 1 mM HFFA+ 100 μM UDCA). UDCA, ursodeoxycholic acid; HFFA, high free fatty acid; ROS, reactive oxygen species; SREBP-1c, sterol regulatory element-binding protein-1c; CD36, cluster of differentiation 36; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; FXR, farnesoid X receptor; Fas , fatty acid synthase; Scd-1, stearoyl-CoA desaturase-1; qRT-PCR, quantitative real-time polymerase chain reaction; Gapdh , glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Alpha mouse liver 12 (AML12) cell line (passages 7–10, obtained from Bioresource Collection and Research Centre, Taiwan) was grown at 37 °C in Dulbecco’s modified Eagle’s medium/Ham’s nutrient mixture F-12 (DMEM/F12), 10% fetal bovine serum (FBS), 2 mmol/L l -Glutamine and 100 μg/mL penicillin and streptomycin (both from Gibco, AntiSel, Greece) at 5% CO 2 .

Techniques: Staining, Quantitative RT-PCR, Expressing, Immunofluorescence, Binding Assay, Real-time Polymerase Chain Reaction

Journal: Nature Communications

Article Title: Editing DNA methylation in vivo

doi: 10.1038/s41467-025-67222-5

Figure Lengend Snippet: A Schematic representation of the Lox-Stop-Lox-dCas9-DNMT3A-P2A-GFP (LSL-dC9-D) transgene cassette inserted at the Rosa26 locus. pCAG cytomegalovirus enhancer fused with chicken beta-actin promoter and rabbit beta-globin splice acceptor, LSL Lox-stop-lox cassette, NLS nuclear localization sequence, P2A porcine teschivoris-1 2A self-cleaving sequence, eGFP enhanced green fluorescent protein, WPRE woodchuck hepatitis virus posttranscriptional regulatory element, bGHpA bovine growth hormone polyadenylation signal. B Western blot of DNMT3A, dCas9, and Tubulin expressions in brain tissue isolated from LSL-dCas9-DNMT3A-GFP mice and LSL-dCas9-DNMT3A-GFP; EIIa-Cre mice. C Immunofluorescent staining of GFP in the hippocampus of LSL-dCas9-DNMT3A-GFP and LSL-dCas9-DNMT3A-GFP; EIIa-Cre mice. Scale bar: 100 μm. D Immunofluorescent staining of DAPI, mCherry, GFP, dCas9 colocalization in mice injected contralaterally with either AAV9-mCherry or AAV9-mCherry-Cre. Scale bar: 100 μm. E Quantification of dCas9-DNMT3A induction efficiency in mCherry and mCherry-Cre labeled cells. ( n = 3 mice per group, two-sided t test, P = 0.000010).

Article Snippet: The doxycycline-inducible dCas9-DNMT3A AML12 cell line was constructed by transfecting wild-type AML12 cells with a piggyBac transposase constructs 137 and 138-dCas9-DNMT3a (Addgene 84570) and selecting with hygromycin for 7 days.

Techniques: Sequencing, Virus, Western Blot, Isolation, Staining, Injection, Labeling

Journal: Nature Communications

Article Title: Editing DNA methylation in vivo

doi: 10.1038/s41467-025-67222-5

Figure Lengend Snippet: A Schematic of designed sgRNAs targeting the Pcsk9 promoter and Pyro-seq assay area in yellow. B Pcsk9 expression in AML12 cells after DNA methylation editing by dCas9-DNMT3A with sgRNAs in ( A ). ( n = 5 biological replicates per group, one way ANOVA (Dunnett’s test vs Scr), sgRNA-2 vs Scr P = 0.0058, sgRNA-1 + 2 vs Scr P = 0.00147). C DNA methylation status of the Pcsk9 promoter after DNA methylation editing by dCas9-DNMT3A in AML12 cells measured by pyrosequencing. Pcsk9 sgRNAs refers to editing with sgRNAs 1 + 2. ( n = 3 biological replicates per group, one-way ANOVA with Tukey’s multiple comparisons per CpG, * P < 0.05, exact P -values provided in source data). D Scheme for in vivo repression of Pcsk9 by targeted methylation of the Pcsk9 promoter in the liver. Created in BioRender. Liu, S. (2025) https://BioRender.com/m5iikln . E DNA methylation status of the Pcsk9 promoter after 6 weeks from liver tissue isolated from dCas9-DNMT3A mice injected with AAV containing Cre and Scr or Pcsk9 targeting sgRNAs measured by pyrosequencing of area ( A and B ). ( n = 3 (PBS), n = 9 (Scr), n = 10 ( Pcsk9 ) mice, one-way ANOVA (Tukey’s test exact P values in source data). F Pcsk9 transcript expression in livers from ( D ). ( n = 5 (Scr) and n = 6 ( Pcsk9 ) mice, two-sided t test, P = 0.0292). G Western blot of LDLR and PCSK9 protein expression levels in liver tissue in ( D ). H Quantification of LDLR and PCSK9 protein levels from western blot in ( G ). ( n = 6 (Scr) and n = 7 ( Pcsk9 ) mice, two-sided t test, LDLR P = 0.0292, PCSK9 P = 0.040). I Representative immunofluorescent staining of LDLR in livers from Scr and Pcsk9 targeted livers. Scale bar: 50 μm. J Quantification of % area stained of LDLR in livers from Scr and Pcsk9 targeted livers. ( n = 4 mice per group, two-sided t test, P = 0.0093). K Serum LDL cholesterol measured biweekly in dCas9-DNMT3A mice injected with Scr or Pcsk9 targeting sgRNAs. ( n = 9 mice per group, two-way ANOVA repeated measures, P = 0.000021). L Quantification of serum PCSK9 levels at study endpoint from Scr and Pcsk9 targeted livers. ( n = 5 (Scr) and n = 6 ( Pcsk9 ) mice, two-sided t test, Absolute p = 0.0032, Relative p = 0.00078).

Article Snippet: The doxycycline-inducible dCas9-DNMT3A AML12 cell line was constructed by transfecting wild-type AML12 cells with a piggyBac transposase constructs 137 and 138-dCas9-DNMT3a (Addgene 84570) and selecting with hygromycin for 7 days.

Techniques: Expressing, DNA Methylation Assay, In Vivo, Methylation, Isolation, Injection, Western Blot, Staining

Journal: Nature Communications

Article Title: Editing DNA methylation in vivo

doi: 10.1038/s41467-025-67222-5

Figure Lengend Snippet: A IGV browser track of anti-Cas9 ChIP-seq data demonstrating the specific binding of dCas9-DNMT3A to the Pcsk9 promoter in DNA methylation edited AML cells. B Representative IGV track of WGBS data of Scr and Pcsk9 sgRNA injected livers at the Pcsk9 locus. The differentially methylated region (DMR) at the Pcsk9 promoter is highlighted in red. C Quantification of average methylation at the DMR identified at the Pcsk9 promoter in Scr and Pcsk9 targeted livers. D DNA methylation of the dCas9-DNMT3A binding sites in Scr and Pcsk9 targeted livers measured by whole genome bisulfite sequencing (WGBS). Pcsk9 is labeled by a red dot and other sites are labeled by black dots. The dashed lines represent a 20% change of DNA methylation level. E RNA-seq of Scr sgRNA and Pcsk9 sgRNA injected livers. Pcsk9 is labeled by a red dot and other genes associated with dCas9-TET1 binding sites are labeled by black dots. The dashed lines represent a twofold change in expression between the conditions.

Article Snippet: The doxycycline-inducible dCas9-DNMT3A AML12 cell line was constructed by transfecting wild-type AML12 cells with a piggyBac transposase constructs 137 and 138-dCas9-DNMT3a (Addgene 84570) and selecting with hygromycin for 7 days.

Techniques: ChIP-sequencing, Binding Assay, DNA Methylation Assay, Injection, Methylation, Methylation Sequencing, Labeling, RNA Sequencing, Expressing