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: Autophagy

Article Title: AMPK protects against alcohol-induced liver injury through UQCRC2 to up-regulate mitophagy

doi: 10.1080/15548627.2021.1886829

Figure Lengend Snippet: AMPK attenuates alcohol-induced liver injury and mitochondrial injury. ( A-C, F ) Analysis of mice on pair-fed mice (n = 15), 10-d eth-fed (n = 15), or treated with metformin at 150 mg/kg/day (Eth-fed+met, n = 15). ( D, E, G ) Analysis of AML-12 cells on control, 200 mM ethanol-treated for 24 h (Eth), 200 mM ethanol + 3.2 mM metformin-treated for 24 h (Eth+ Met). (A) Electron microscopy analyses of mice liver display mitochondria with normal morphology, destroyed cristae structure, and fewer mitochondrial number. (B) Quantification of mitochondrial number in 200 μm 2 (scale bar: 1 μm). (C) qRT-PCR was used to identify mtDNA level in mice serum, which can indicate mitochondrial injury level in mice hepatocytes. (D) AML-12 cells were stained with MTRC to identify the relative mtROS level. The fluorescence light intensity was measured by a multi-function micro reader. (E) AML-12 cells were stained with MTRC, and images were captured with a fluorescence microscope (scale bar: 50 μm) Arrow indicates mtROS points. (F, G) Western blot results show LC3-II:LC3-I ratio and SQSTM1 level in mice liver and AML-12 cells. ACTB was used as an internal reference in whole-cell lysates. Each experiment was repeated 3 times. Data are expressed as the mean ± SEM. * p < 0.05, ** p < 0.01

Article Snippet: For other experiments, AML-12 cells were also treated with CCCP (MCE, HY-100,941; 10 μM), as shown in figure legends.

Techniques: Control, Electron Microscopy, Quantitative RT-PCR, Staining, Fluorescence, Microscopy, Western Blot

Journal: Autophagy

Article Title: AMPK protects against alcohol-induced liver injury through UQCRC2 to up-regulate mitophagy

doi: 10.1080/15548627.2021.1886829

Figure Lengend Snippet: AMPK upregulates mitophagy level in mice and AML-12 cells. ( A-C, G ) Analysis of AML-12 cells on control, 200 mM ethanol-treated for 24 h (Eth), 200 mM ethanol + 3.2 mM metformin-treated for 24 h (Eth+ Met). (D-F) Analysis of mice on pair-fed mice (n = 15), 10-d eth-fed (n = 15), or treated with metformin at 150 mg/kg/day (Eth-fed+met, n = 15). (A) AML-12 cells were transfected with mito timer plasmid in advance (6 h), and were treated with ethanol, metformin (24 h). A multi-function micro reader was used to detect the 500:583 nm (green:red) fluorescence signal ratio. (B) AML-12 cells were treated with ethanol, metformin for 24 h, and were stained with MTDR for 15 min. A multi-function micro reader was used to detect 665 nm fluorescence light intensity. (C) FCM (flow cytometry) experiments and analysis. AML-12 cells were transfected with mtKeima plasmid in advance (6 h), and were treated with ethanol, metformin (24 h). Analysis of mitophagic ratio in Con, Eth, and Eth+Met cells. (D) IF staining results of mitochondria (TOMM20, red), LC3 (green) and nucleus (DAPI, blue) Scale bar: 10 μm. (E) Count of cells with LC3 on mitochondria (merged signal, yellow) in 200 μm 2 . (F, G) Western blot results show the protein (PINK1, PRKN) level in mitochondrial lysates of mice livers and AML-12 cells. TOMM20 was used as an internal reference in mitochondrial lysates. Each experiment was repeated 3 times. Data are expressed as the mean ± SEM. * p < 0.05, ** p < 0.01

Article Snippet: For other experiments, AML-12 cells were also treated with CCCP (MCE, HY-100,941; 10 μM), as shown in figure legends.

Techniques: Control, Transfection, Plasmid Preparation, Fluorescence, Staining, Flow Cytometry, Western Blot

Journal: Autophagy

Article Title: AMPK protects against alcohol-induced liver injury through UQCRC2 to up-regulate mitophagy

doi: 10.1080/15548627.2021.1886829

Figure Lengend Snippet: AMPK upregulates UQCRC2 protein level (Complex III activity) in ALD. (A, C) Analysis of mice on pair-fed mice (n = 15), 10-d eth-fed (n = 15), or treated with metformin at 150 mg/kg/day (Eth-fed+met, n = 15). (B, D) Analysis of AML-12 cells on control, 200 mM ethanol (Eth), 200 mM ethanol + 3.2 mM metformin (Eth+ Met). (A, B) Western blot results: Ethanol caused the low expression of UQCRC2 protein in liver and AML-12 cells, whereas metformin can significantly enhance UQCRC2 protein level. (C, D) Mitochondrial activity assay: Ethanol reduced complex III activity, whereas metformin can upregulate complex III activity in 10 days ethanol-fed mice or ethanol-treated AML-12 cells. (E) IHC results of UQCRC2 in human samples, and IHC scores analysis. Each experiment was repeated 3 times. Data are expressed as the mean ± SEM. * p < 0.05, ** p < 0.01

Article Snippet: For other experiments, AML-12 cells were also treated with CCCP (MCE, HY-100,941; 10 μM), as shown in figure legends.

Techniques: Activity Assay, Control, Western Blot, Expressing

Journal: Autophagy

Article Title: AMPK protects against alcohol-induced liver injury through UQCRC2 to up-regulate mitophagy

doi: 10.1080/15548627.2021.1886829

Figure Lengend Snippet: AMPK indirectly regulates UQCRC2. (A) qRT-PCR results of Uqcrc2 mRNA fold in pair-fed mice (n = 15), 10-d eth-fed (n = 15), or treated with metformin at 150 mg/kg/day (Eth-fed+met, n = 15). (B) A heatmap showed differentially expressed RNA with the same expression pattern in pair-fed and 10-d eth-fed mice. (C) Nfe2l2 fold change (RNA-seq results). (D) Gene promoter analysis: Three sites in Uqcrc2 promoter that NFE2L2 can bind. (E) Luciferase reporter gene results in AML-12 cells. AML-12 cells were transfected with NC 1 (pGL3-basic) + NC 2 (pcDNA3.1), NC 1 + pGL3- Nfe2l2 , NC 2 + pcDNA3.1- Uqcrc2 , pGL3- Nfe2l2 + pcDNA3.1- Uqcrc2 . Data are presented as the ratio of Renilla luciferase activity to firefly activity (n = 3). (F) qRT-PCR results of Nfe2l2 mRNA fold in pair-fed mice (n = 15), 10-d eth-fed (n = 15), or treated with metformin at 150 mg/kg/day (Eth-fed+met, n = 15). (G) ChIP assay showing the interaction of NFE2L2 with the potential binding site on the Uqcrc2 promoter in AML-12 cells. Metformin enhanced UQCRC2 fold number in the anti- NFE2L2 group, whereas shPrkaa1 reduced metformin-induced high enrichment of UQCRC2 in the anti-NFE2L2 group. (H, I) Analysis of AML-12 cells on NC (negative control), NC+ 3.2 Metformin (Met), 3.2 mM Met + shPrkaa1 (50 nM), NC+ ethanol, NC+ethanol + metformin, Ethanol + Metformin + shPrkaa1 . Western blot results show NFE2L2 and UQCRC2 protein levels in AML-12 cells. ACTB was used as an internal reference in whole-cell lysates. Each experiment was repeated 3 times. Data are expressed as the mean ± SEM. * p < 0.05, ** p < 0.01

Article Snippet: For other experiments, AML-12 cells were also treated with CCCP (MCE, HY-100,941; 10 μM), as shown in figure legends.

Techniques: Quantitative RT-PCR, Expressing, RNA Sequencing, Luciferase, Transfection, Activity Assay, Binding Assay, Negative Control, Western Blot

Journal: Autophagy

Article Title: AMPK protects against alcohol-induced liver injury through UQCRC2 to up-regulate mitophagy

doi: 10.1080/15548627.2021.1886829

Figure Lengend Snippet: The effects of UQCRC2 genetic intervention in mice and AML-12 cells. ( A- E, I- K ) Analysis of mice on pair-fed mice was injected with AAV-NC or lentivirus-NC (n = 15), pair-fed mice were injected with AAV-UQCRC2 OE (n = 15), or pair-fed mice were injected with AAV-UQCRC2 KD (n = 15). (F-H) Analysis of AML-12 cells on NC plasmid (50 nM), UQCRC2 OE plasmid (50 nM), UQCRC2 KD plasmid (50 nM). (A, B) Test of GPT, and GOT1 levels in serum. (C) HE staining results and analysis: UQCRC2 OE or KD cannot cause liver injury (scale bar: 200 μm). (D) Oil red staining results and analysis: UQCRC2 OE or KD cannot influence lipid accumulation in mice liver (scale bar: 100 μm). (E). qRT-PCR was used to identify the mtDNA level in mice serum. (F) AML-12 cells were stained with MTRC to identify the relative mtROS level. The fluorescence light intensity was measured by a multi-function microplate reader. (G) AML-12 cells were treated with ethanol, metformin for 24 h, and were stained with MTDR for 15 min. A multi-function micro reader was used to detect 665 nm fluorescence light intensity. (H) FCM (flow cytometry) experiments and analysis. AML-12 cells were transfected with mtKeima plasmid in advance (6 h), and were treated with ethanol, metformin (24 h). Analysis of mitophagic ratio in NC, UQCRC2 OE, and UQCRC2 KD cells. (I) IF staining results of mitochondria (TOMM20, red), LC3 (green) and nucleus (DAPI, blue) Scale bar: 10 μm. Count of cells with LC3 on mitochondria (merged signal, yellow) in 200 μm 2 . (J, K) Western blot results show the protein (PINK1, PRKN) level in mice livers. TOMM20 was used as an internal reference in mitochondrial lysates. WB results show LC3-II:LC3-I ratio in mice liver. ACTB was used as an internal reference in whole-cell lysates. Each experiment was repeated 3 times. Data are expressed as the mean ± SEM. * p < 0.05, ** p < 0.01

Article Snippet: For other experiments, AML-12 cells were also treated with CCCP (MCE, HY-100,941; 10 μM), as shown in figure legends.

Techniques: Injection, Plasmid Preparation, Staining, Quantitative RT-PCR, Fluorescence, Flow Cytometry, Transfection, Western Blot

Journal: Autophagy

Article Title: AMPK protects against alcohol-induced liver injury through UQCRC2 to up-regulate mitophagy

doi: 10.1080/15548627.2021.1886829

Figure Lengend Snippet: UQCRC2 intervention regulates mitophagy in AML-12 cells. (A) OCR in NC and UQCRC2 KD AML-12 cells. (B) ATP production level in NC and UQCRC2 KD AML-12 cells. (C) JC-1 staining results of NC and UQCRC2 KD AML-12 cells. The ratio of absorbance 585:529 is ΔΨ. (D) OCR in NC, 10 μM CCCP, and 10 μM CCCP+UQCRC2 KD AML-12 cells. (E) ATP production level in NC, 10 μM CCCP, and 10 μM CCCP+UQCRC2 KD AML-12 cells. (F) Western blot results show the LC3 II/LC3 I ratio in AML-12 cells. ACTB was used as an internal reference in whole-cell lysates. (G) Western blot results show the p-AMPK level in NC and UQCRC2 KD AML-12 cells. ACTB was used as an internal reference in whole-cell lysates. (H) Western blot results show the PINK1 level in NC and PINK1 KD AML-12 cells, (I) and PRKN protein level on mitochondria in NC+CCCP, PINK1 KD+CCCP, and PINK1 KD+ UQCRC2 OE+CCCP AML-12 cells. ACTB was used as an internal reference in whole-cell lysates. TOMM20 was used as an internal reference in mitochondrial lysates. Each experiment was repeated 3 times. Data are expressed as the mean ± SEM. * p < 0.05, ** p < 0.01

Article Snippet: For other experiments, AML-12 cells were also treated with CCCP (MCE, HY-100,941; 10 μM), as shown in figure legends.

Techniques: Staining, Western Blot

Journal: Autophagy

Article Title: AMPK protects against alcohol-induced liver injury through UQCRC2 to up-regulate mitophagy

doi: 10.1080/15548627.2021.1886829

Figure Lengend Snippet: UQCRC2 OE or KD can attenuate or deteriorate ALD through mitophagy. ( A-E, H, I, K ) Analysis of mice on 10-d Eth-fed mice was injected with AAV-NC or lentivirus-NC (n = 15), 10-d Eth-fed mice were injected with AAV-UQCRC2 OE (n = 15), or 10-d Eth-fed were injected with AAV-UQCRC2 KD (n = 15). ( F, G, J ) Analysis of AML-12 cells on 200 mM Eth + NC plasmid (50 nM), Eth+ UQCRC2 OE plasmid (50 nM), Eth+ UQCRC2 KD plasmid (50 nM). (A, B) Test of GPT, and GOT1 level in serum. (C) HE staining results and analysis (scale bar: 200 μm). (D) Oil red staining results and analysis (scale bar: 100 μm). (E) qRT-PCR was used to identify the mtDNA level in mice serum. (F) AML-12 cells were stained with MTRC to identify the relative mtROS level. The fluorescence light intensity was measured by a multi-function micro reader. (G) AML-12 cells were treated were stained with MTDR for 15 min. A multi-function micro reader was used to detect 665 nm fluorescence light intensity. (H, I) IF staining results of mitochondria (TOMM20, red), LC3 (green) and nucleus (DAPI, blue) Scale bar: 10 μm. Count of cells with LC3 on mitochondria (merged signal, yellow) in 200 μm 2 . (J) FCM (flow cytometry) experiments and analysis. AML-12 cells were transfected with mtKeima plasmid in advance (6 h), and were detected by FCM. Analysis of mitophagic ratio in NC+Eth, Eth+UQCRC2 OE, and Eth+UQCRC2 KD cells. (K) Western blot results showing the protein (PINK1, PRKN) level in mice livers. TOMM20 was used as an internal reference in mitochondrial lysates. Western blot results show LC3-II:LC3-I ratio in mice liver. ACTB was used as an internal reference in whole-cell lysates. Each experiment was repeated 3 times. Data are expressed as the mean ± SEM. * p < 0.05, ** p < 0.01

Article Snippet: For other experiments, AML-12 cells were also treated with CCCP (MCE, HY-100,941; 10 μM), as shown in figure legends.

Techniques: Injection, Plasmid Preparation, Staining, Quantitative RT-PCR, Fluorescence, Flow Cytometry, Transfection, Western Blot

Journal: Autophagy

Article Title: AMPK protects against alcohol-induced liver injury through UQCRC2 to up-regulate mitophagy

doi: 10.1080/15548627.2021.1886829

Figure Lengend Snippet: UQCRC2 protein is required for AMPK regulating mitophagy in ALD. ( A-E, I-K ) Analysis of mice on 10 d Eth-fed mice was injected with AAV-NC (n = 15), 10-d Eth-fed mice were orally administrated with 150 mg/kg metformin (n = 15), or 10-d Eth-fed were orally administrated with 150 mg/kg metformin and injected with AAV-UQCRC2 KD (n = 15). (F-H) Analysis of AML-12 cells on 200 mM Eth + NC plasmid (50 nM), Eth+ NC plasmid + 3.2 mM metformin, Eth+ UQCRC2 KD plasmid (50 nM) +metformin. (A, B) Test of GPT and GOT1 level in serum. (C) HE staining results and analysis (scale bar: 200 μm). (D) Oil red staining results and analysis (scale bar: 100 μm). (E) qRT-PCR was used to identify the mtDNA level in mice serum. (F) AML-12 cells were stained with MTRC to identify the relative mtROS level. The fluorescence light intensity was measured by a multi-function micro reader. (G) AML-12 cells were treated were stained with MTDR for 15 min, and were measured by a multi-function microplate reader. (H) FCM (flow cytometry) experiments and analysis. Analysis of mitophagic ratio in AML-12 cells. (I) IF staining results of mitochondria (TOMM20, red), LC3 (green) and nucleus (DAPI, blue) Scale bar: 10 μm. Count of cells with LC3 on mitochondria (merged signal, yellow) in 200 μm 2 . (J, K) Western blot results showing the protein (PINK1, PRKN) level in mice livers. TOMM20 was used as an internal reference in mitochondrial lysates. WB results show LC3-II:LC3-I ratio in mice liver. ACTB was used as an internal reference in whole-cell lysates. Each experiment was repeated 3 times. Data are expressed as the mean ± SEM. * p < 0.05, ** p < 0.01

Article Snippet: For other experiments, AML-12 cells were also treated with CCCP (MCE, HY-100,941; 10 μM), as shown in figure legends.

Techniques: Injection, Plasmid Preparation, Staining, Quantitative RT-PCR, Fluorescence, Flow Cytometry, Western Blot

Journal: Autophagy

Article Title: AMPK protects against alcohol-induced liver injury through UQCRC2 to up-regulate mitophagy

doi: 10.1080/15548627.2021.1886829

Figure Lengend Snippet: PRKN protein is downstream of UQCRC2 in the mitophagy signaling pathway. ( A-E, H-J ) Analysis of mice on 10-d Eth-fed mice was injected with AAV-NC(n = 15), 10-d Eth-fed mice were injected with AAV-UQCRC2 KD (n = 15), or 10-d Eth-fed mice were injected with AAV-UQCRC2 KD and AAV- PRKN OE (n = 15). ( F, G, J ) Analysis of AML-12 cells on 200 mM Eth, Eth+ UQCRC2 KD plasmid (50 nM) + PRKN OE plasmid (50 nM). (A, B). Test of GPT, and GOT1 level in serum. (C). HE staining results and analysis (scale bar: 200 μm). (D). Oil red staining results and analysis (scale bar: 100 μm). (E). qRT-PCR was used to identify the mtDNA level in mice serum. (F). AML-12 cells were stained with MTRC to identify the relative mtROS level. (G). AML-12 cells were treated were stained with MTDR for 15 min, and were measured by micro fluorescence reader at 665 nm. (H, I) IF staining results of mitochondria (TOMM20, red), LC3 (green) and nucleus (DAPI, blue) Scale bar: 10 μm. Count of cells with LC3 on mitochondria (merged signal, yellow) in 200 μm 2 . (J). WB results show LC3-II:LC3-I ratio in mice liver and AML-12 cells. ACTB was used as an internal reference in whole-cell lysates. Each experiment was repeated 3 times. Data are expressed as the mean ± SEM. * p < 0.05, ** p < 0.01

Article Snippet: For other experiments, AML-12 cells were also treated with CCCP (MCE, HY-100,941; 10 μM), as shown in figure legends.

Techniques: Injection, Plasmid Preparation, Staining, Quantitative RT-PCR, Fluorescence

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