Journal: Cells

Article Title: Inhibition of Mitochondrial Complex Function—The Hepatotoxicity Mechanism of Emodin Based on Quantitative Proteomic Analyses

doi: 10.3390/cells8030263

Figure Lengend Snippet: The differentially expressed proteins involved in the oxidative phosphorylation pathway and respiratory chain complex.

Article Snippet: Caspase-8, caspase-3, Ndufs1, Cox7A2, ATP6, SDHA, and Bax inhibitor 1 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques:

Journal: Cell reports

Article Title: Glioblastoma Cell Resistance to EGFR and MET Inhibition Can Be Overcome via Blockade of FGFR-SPRY2 Bypass Signaling

doi: 10.1016/j.celrep.2020.02.014

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: pNB777 , ( Mazo-Vargas et al., 2014 ) , Addgene #60153.

Techniques: Subcloning, Recombinant, Chromatin Immunoprecipitation, Ab Array, Enzyme-linked Immunosorbent Assay, Proliferation Assay, Bicinchoninic Acid Protein Assay, Stable Transfection, Expressing, shRNA, Sequencing, Software

Journal: The Journal of Clinical Investigation

Article Title: ALKBH1-demethylated DNA N 6 -methyladenine modification triggers vascular calcification via osteogenic reprogramming in chronic kidney disease

doi: 10.1172/JCI146985

Figure Lengend Snippet: (A) Leukocyte DNA 6mA level in patients with CKD with (VC, n = 106) or without (non-VC, n = 67) aortic arch calcification. (B and C) Leukocyte DNA 6mA level in subgroups defined by calcification Agatston score (B, n = 67 for non-VC; n = 61 for mild; n = 45 for severe) and Volume score (C, n = 67 for non-VC; n = 53 for mild; n = 53 for severe). (D) Correlation between leukocyte DNA 6mA level and calcification Agatston score from patients with CKD with aortic arch calcification (n = 106). (E) Quantitative real-time PCR analysis of ALKBH1 and N6AMT1 mRNA expression in leukocytes from patients with CKD with (VC, n = 40) or without (non-VC, n = 38) aortic arch calcification. (F and G) Leukocyte ALKBH1 mRNA expression level in subgroups defined by calcification Agatston score (F, n = 38 for non-VC; n = 21 for mild; n = 19 for severe) and Volume score (G, n = 38 for non-VC; n = 13 for mild; n = 27 for severe). (H) Scatterdot plot of correlation between leukocyte ALKBH1 mRNA expression level and calcification Agatston score from patients with CKD with aortic arch calcification (n = 40). (I–K) Representative immunofluorescence pictures (I) and quantification (J) and Western blot analysis (K) of ALKBH1, N6AMT1, and 6mA in radial arteries from CKD (n = 4) and control (n = 4) groups. Scale bars: 50 μm. Statistical significance was assessed using 2-tailed t tests (A, E, J, and K), 1-way ANOVA followed by Bonferroni’s test (B, C, F, and G), and Pearson’s correlation coefficient analysis (D and H). All values are presented as mean ± SD. *P < 0.05.

Article Snippet: HASMCs were infected with recombinant lentivirus expressing control shRNA (Santa Cruz Biotechnology, sc-108080), ALKBH1 shRNA (Santa Cruz Biotechnology, sc-60153-V) according to the manufacturer’s instruction.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Immunofluorescence, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: ALKBH1-demethylated DNA N 6 -methyladenine modification triggers vascular calcification via osteogenic reprogramming in chronic kidney disease

doi: 10.1172/JCI146985

Figure Lengend Snippet: (A) Leukocyte DNA 6mA level in mice fed with adenine diet (CKD, n = 13) or normal chow diet (control, n = 12) for 8 weeks. Leukocytes were isolated from peripheral blood. (B and C) Mice leukocyte DNA 6mA level in different subgroups defined by the percentage of calcification lesion size in aortic smooth muscle layer (B, n = 12 for non-VC; n = 6 for mild; n = 7 for severe). Scatter dot plot of correlation between mice leukocyte DNA 6 mA level and percentage of calcification lesion size in aortic smooth muscle layer from mice fed with adenine diet for 8 weeks (C, n = 13). (D) The mRNA expression levels of Alkbh1 and N6amt1 in leukocytes from mice with different diets (n = 12 per group). (E) Representative immunohistochemistry pictures and quantification of ALKBH1, N6AMT1, and 6mA in mice aortic smooth muscle layer (n = 10 for control; n = 12 for CKD). Scale bars: 50 μm. (F) Western blot analysis of ALKBH1 and N6AMT1 expression in mice aortic arch (n = 4 for control; n = 5 for CKD). (G–I) Calcium content (G), Western blot analysis of ALKBH1 and N6AMT1 (H), and DNA 6 mA level (I) in mice aortic rings incubated with osteogenic medium for the indicated time (0, 3, 5, 7, 10, and 14 days) (n = 4–6 per group). Statistical significance was assessed using 2-tailed t tests (A and D–F), 1-way ANOVA followed by Bonferroni’s test (B) or Dunnett’s test (G–I), and Pearson’s correlation coefficient analysis (C). All values are presented as mean ± SD. *P < 0.05 vs. Pi (0 day) in (G–I).

Article Snippet: HASMCs were infected with recombinant lentivirus expressing control shRNA (Santa Cruz Biotechnology, sc-108080), ALKBH1 shRNA (Santa Cruz Biotechnology, sc-60153-V) according to the manufacturer’s instruction.

Techniques: Isolation, Expressing, Immunohistochemistry, Western Blot, Incubation

Journal: The Journal of Clinical Investigation

Article Title: ALKBH1-demethylated DNA N 6 -methyladenine modification triggers vascular calcification via osteogenic reprogramming in chronic kidney disease

doi: 10.1172/JCI146985

Figure Lengend Snippet: (A) Western blot analysis identifying the ALKBH1 deficiency in arteries (n = 6 per group). Mice were injected via tail vein with AAV carrying scrambled shRNA (sh-Scr) or Alkbh1 shRNA (sh-ALKBH1) at 4 weeks after adenine diet and then fed for another 4 weeks. (B–D) Von Kossa staining (B and C) and calcium content quantification of aortic arch (D) were performed in different experimental groups for detecting mineralization (n = 10–12 per group). Scale bar: 100 μm. (E) Photomicrographs of Alizarin red staining in mice primary VSMCs pretransfected with indicated treatment and exposed in osteogenic medium for another 14 days (n = 6 per group). (F and G) Bar graphs representative of calcium content (F) and ALP activity (G) in mice primary VSMCs from all of the experimental cohorts (n = 6 per group). (H) ALKBH1 overexpression in arteries confirmed by Western blot (n = 6 per group). Mice were injected with AAV-Vector or AAV-ALKBH1 at 4 weeks after the adenine diet and then fed for another 4 weeks. (I–K) Percentage of positive von Kossa staining (I and J) and calcium content (K) quantified in the aortic arch from the different cohorts (n = 10–12 per group). Scale bar: 100 μm. (L) Representative images of Alizarin red staining in mice primary VSMCs after indicated transfection and osteogenic medium exposure for another 14 days (n = 6 per group). (M and N) Scatter dot plots representative of calcium content (M) and ALP activity (N) in mice primary VSMCs from all of the experimental cohorts (n = 6 per group). Statistical significance was assessed using 2-tailed t tests (A and H) and 1-way ANOVA followed by Dunnett’s test (C–G, and J–N). All values are presented as mean ± SD. *P < 0.05.

Article Snippet: HASMCs were infected with recombinant lentivirus expressing control shRNA (Santa Cruz Biotechnology, sc-108080), ALKBH1 shRNA (Santa Cruz Biotechnology, sc-60153-V) according to the manufacturer’s instruction.

Techniques: Western Blot, Injection, shRNA, Staining, Activity Assay, Over Expression, Plasmid Preparation, Transfection

Journal: The Journal of Clinical Investigation

Article Title: ALKBH1-demethylated DNA N 6 -methyladenine modification triggers vascular calcification via osteogenic reprogramming in chronic kidney disease

doi: 10.1172/JCI146985

Figure Lengend Snippet: (A) Western blot analysis of osteogenic phenotype marker (OPN, OCN, and Collagen I) and contractile phenotype marker (SM22α, α-SMA, and Calponin1) expression in mice primary VSMCs cultured in osteogenic medium for 14 days. (B) Quantitative real-time PCR analysis of Alkbh1 expression in mice primary VSMCs, which were pretransfected with AAV encoding scrambled or Alkbh1 shRNA for 48 hours, and then cultured in osteogenic medium for another 14 days. (C) Quantitative DNA 6mA level in ALKBH1-deficient mice primary VSMCs. (D) Quantitative real-time PCR analysis of Alkbh1 expression in mice primary VSMCs, which were preinfected with AAV-Vector or AAV-ALKBH1 for 48 hours, and then cultured in osteogenic medium for another 14 days. (E) Quantitative DNA 6mA level in ALKBH1-overexpressed mice primary VSMCs. (F) Western blot analysis of osteogenic phenotype marker and contractile phenotype marker expression in mice primary VSMCs with ALKBH1 depletion. (G) Western blot analysis of osteogenic phenotype marker and contractile phenotype marker expression in mice primary VSMCs with ALKBH1 overexpression. Statistical significance was assessed using 1-way ANOVA followed by Dunnett’s test. n = 4–6 for each group. All values are presented as mean ± SD. *P < 0.05 vs. Pi (0 day) in (A).

Article Snippet: HASMCs were infected with recombinant lentivirus expressing control shRNA (Santa Cruz Biotechnology, sc-108080), ALKBH1 shRNA (Santa Cruz Biotechnology, sc-60153-V) according to the manufacturer’s instruction.

Techniques: Western Blot, Marker, Expressing, Cell Culture, Real-time Polymerase Chain Reaction, shRNA, Plasmid Preparation, Over Expression

Journal: The Journal of Clinical Investigation

Article Title: ALKBH1-demethylated DNA N 6 -methyladenine modification triggers vascular calcification via osteogenic reprogramming in chronic kidney disease

doi: 10.1172/JCI146985

Figure Lengend Snippet: (A) Western blot analysis of ALKBH1, BMP2, RUNX2, SOX9, and DLX5 expression in calcified mice primary VSMCs with AAV sh-Scr or AAV sh-ALKBH1 transfection (n = 4 per group). (B and C) Representative immunofluorescence images (B) and quantification (C) of α-SMA and BMP2 costained in aortas from indicated experimental cohorts (n = 6 per group). Scale bar: 50 μm. (D) Western blot analysis of osteogenic phenotype marker (RUNX2, OPN, OCN, and Collagen I) expression in mice primary VSMCs, which preinfected with AAV sh-Scr or AAV sh-BMP2 together with AAV-Vector or AAV-ALKBH1 and then incubated in calcifying medium for another 14 days. (E and F) Alizarin red staining (E) and ALP activity assay (F) performed in all of the groups for detecting calcification formation (n = 4–5 per group). (G) Quantification of calcium content in mice aortic ring cultured in calcifying medium with indicated transfection (n = 5 per group). Statistical significance was assessed using 1-way ANOVA followed by Dunnett’s test (A–C) or Bonferroni’s test (D–G). All values are presented as mean ± SD. *P < 0.05.

Article Snippet: HASMCs were infected with recombinant lentivirus expressing control shRNA (Santa Cruz Biotechnology, sc-108080), ALKBH1 shRNA (Santa Cruz Biotechnology, sc-60153-V) according to the manufacturer’s instruction.

Techniques: Western Blot, Expressing, Transfection, Marker, Plasmid Preparation, Incubation, Staining, ALP Activity Assay, Cell Culture

Journal: The Journal of Clinical Investigation

Article Title: ALKBH1-demethylated DNA N 6 -methyladenine modification triggers vascular calcification via osteogenic reprogramming in chronic kidney disease

doi: 10.1172/JCI146985

Figure Lengend Snippet: (A) Quantitative real-time PCR analysis of Bmp2 expression in primary mice VSMCs with ALKBH1 depletion (n = 6 per group). (B and C) Quantitative real-time PCR analysis of Bmp2 expression in the aortic arch from mice with ALKBH1 knockdown (B) or ALKBH1 overexpression (C) (n = 12 per group). (D) Quantitative real-time PCR analysis of Bmp2 expression in mice primary VSMCs treated with actinomycin D (5 mg/mL) for a different time after AAV sh-Scr or AAV sh-ALKBH1 transfection (n = 3 per group). Gene expression was normalized to Gapdh. Statistical significance was assessed using 1-way ANOVA followed by Dunnett’s test. All values are presented as mean ± SD. *P < 0.05.

Article Snippet: HASMCs were infected with recombinant lentivirus expressing control shRNA (Santa Cruz Biotechnology, sc-108080), ALKBH1 shRNA (Santa Cruz Biotechnology, sc-60153-V) according to the manufacturer’s instruction.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Over Expression, Transfection

Journal: The Journal of Clinical Investigation

Article Title: ALKBH1-demethylated DNA N 6 -methyladenine modification triggers vascular calcification via osteogenic reprogramming in chronic kidney disease

doi: 10.1172/JCI146985

Figure Lengend Snippet: (A) Integrative genomics viewer plots showing the increasing 6mA peaks (selected one marked as ChIP1-3) in human BMP2 gene (hg19) region with ALKBH1 knockdown via shRNA lentiviral constructs. (B) ChIP-qPCR assay displaying the 6mA enrichment on the 3 BMP2 fragments in treated HASMCs (n = 4 per group). (C and D) Quantitative Western blot (C) and real-time PCR analysis of BMP2 expression (D) in HASMCs with scramble or OCT4 siRNA transfection under calcifying condition (n = 3 per group). (E) ChIP-qPCR assay with Oct4 or IgG antibody for the ChIP-1 enrichment in treated HASMCs (n = 4 per group). (F) Western blot analysis of Oct4 in HASMCs incubated with osteogenic medium after transfection with scrambled siRNA (si-Scr) or ALKBH1 siRNA (si-ALKBH1). (G) Logos of the standard Oct4 motif and schematic of human BMP2 promoter showing wide-type (WT) and deleted (Del) binding sites for Oct4 within the first 6mA peak. (H) Bar graphs representative of the luciferase activity analyzed in HASMCs after cotransfection with control Renilla luciferase plasmid and serial deletion constructs of BMP2 promoter-driven luciferase reporters containing WT or Del Oct4 site (n = 5 per group). (I) Relative promoter activities measured by dual-luciferase reporter assay in HASMCs, which pretreated with indicated siRNA and then infected with pGL3-Oct4-WT or pGL3-Oct4-Del under calcifying conditions (n = 4 per group). Statistical significance was assessed using 1-way ANOVA followed by Bonferroni’s test. All values are presented as mean ± SD. *P < 0.05.

Article Snippet: HASMCs were infected with recombinant lentivirus expressing control shRNA (Santa Cruz Biotechnology, sc-108080), ALKBH1 shRNA (Santa Cruz Biotechnology, sc-60153-V) according to the manufacturer’s instruction.

Techniques: shRNA, Construct, Western Blot, Real-time Polymerase Chain Reaction, Expressing, Transfection, Incubation, Binding Assay, Luciferase, Activity Assay, Cotransfection, Plasmid Preparation, Reporter Assay, Infection

Journal: The Journal of Clinical Investigation

Article Title: ALKBH1-demethylated DNA N 6 -methyladenine modification triggers vascular calcification via osteogenic reprogramming in chronic kidney disease

doi: 10.1172/JCI146985

Figure Lengend Snippet: (A–C) Representative von Kossa staining images (A) and quantification (B) of aortic rings from Oct4WT/WT-Myh11-Cre/ERT2 (WT) and Oct4F/F-Myh11-Cre/ERT2 (Oct4–/–) mice cultured in osteogenic medium for 14 days. Bar graphs representative of calcium content (C) from these 2 groups (n = 6 per group). Scale bar: 100 μm. (D–G) Western blot analysis (D) of BMP2, Oct4, ALKBH1, and osteogenic phenotype marker (OCN and Collagen I) expression in calcified primary VSMCs from WT or Oct4–/– mice transfected with AAV-Vector or AAV-ALKBH1. Quantitative real-time PCR analysis of Bmp2 expression in all of the experimental cohorts (E). Alizarin red staining (F) and calcium content quantification (G) performed in all of the groups for detecting calcification formation (n = 4–6 per group). Statistical significance was assessed using 2-tailed t tests (B and C) and 1-way ANOVA followed by Bonferroni’s test (D–G). All values are presented as mean ± SD. *P < 0.05.

Article Snippet: HASMCs were infected with recombinant lentivirus expressing control shRNA (Santa Cruz Biotechnology, sc-108080), ALKBH1 shRNA (Santa Cruz Biotechnology, sc-60153-V) according to the manufacturer’s instruction.

Techniques: Staining, Cell Culture, Western Blot, Marker, Expressing, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction