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alkbh1 shrna (Santa Cruz Biotechnology)

Name: alkbh1 shrna
Brand: Santa Cruz Biotechnology
Rating: 86 (1 reviews)

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Santa Cruz Biotechnology alkbh1 shrna

(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 <t>ALKBH1</t> 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.

Alkbh1 Shrna, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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alkbh1 shrna - by Bioz Stars, 2025-03

86/100 stars

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1) Product Images from "ALKBH1-demethylated DNA N 6 -methyladenine modification triggers vascular calcification via osteogenic reprogramming in chronic kidney disease"

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

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI146985

Figure Legend 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.

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

(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).

Figure Legend 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).

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

(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.

Figure Legend 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.

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

(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).

Figure Legend 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).

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

(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.

Figure Legend 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.

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

(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.

Figure Legend 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.

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

(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.

Figure Legend 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.

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

(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.

Figure Legend 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.

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

Image Search Results

( a ) ALKBH1 and DENV2 NS5 immunoblots of DENV2 NGC-and mock-infected cells (left). Densitometric quantitation of NS5 protein (right). ( b ) siRNA knockdown of ALKBH1 assessed by immunoblotting. ( c ) LC-MS/MS profiling of tRNA modifications in ALKBH1 knockdown versus control knockdown cells. ( d ) DENV2 NS3 immunoblot analysis in ALKBH1 knockdown versus control knockdown cells (left). Densitometry quantitation of NS3 protein (right). ( e ) New virus particle production in ALKBH1 knockdown versus control knockdown cells determined by plaque assay. ( f ) DENV2 NS3 immunoblot analysis in ALKBH1-overexpressing versus control cells (left). Densitometry quantitation of NS3 protein (right). ( g ) LC-MS/MS profiling of tRNA modifications in ALKBH1-overexpressing versus control cells. ( h ) New virus particle production at 24 hpi in ALKBH1-overexpressing versus control cells determined by plaque assay. Graphs in all panels show mean ±} SD for 3 biological replicates. All statistical significance was determined using Student’s t test, *p<0.05; **p<0.01.

Journal: bioRxiv

Article Title: Dengue virus exploits the host tRNA epitranscriptome to promote viral replication

doi: 10.1101/2023.11.05.565734

Figure Lengend Snippet: ( a ) ALKBH1 and DENV2 NS5 immunoblots of DENV2 NGC-and mock-infected cells (left). Densitometric quantitation of NS5 protein (right). ( b ) siRNA knockdown of ALKBH1 assessed by immunoblotting. ( c ) LC-MS/MS profiling of tRNA modifications in ALKBH1 knockdown versus control knockdown cells. ( d ) DENV2 NS3 immunoblot analysis in ALKBH1 knockdown versus control knockdown cells (left). Densitometry quantitation of NS3 protein (right). ( e ) New virus particle production in ALKBH1 knockdown versus control knockdown cells determined by plaque assay. ( f ) DENV2 NS3 immunoblot analysis in ALKBH1-overexpressing versus control cells (left). Densitometry quantitation of NS3 protein (right). ( g ) LC-MS/MS profiling of tRNA modifications in ALKBH1-overexpressing versus control cells. ( h ) New virus particle production at 24 hpi in ALKBH1-overexpressing versus control cells determined by plaque assay. Graphs in all panels show mean ±} SD for 3 biological replicates. All statistical significance was determined using Student’s t test, *p<0.05; **p<0.01.

Article Snippet: Transfections were performed using 25 nM ALKBH1-specific (8846, Dharmacon) or non-targeting control (D-001810-10-05, Dharmacon) siRNA, 500 ng plasmid DNA and lipofectamine 3000 (Invitrogen) as indicated, according to the manufacturer’s instructions.

Techniques: Western Blot, Infection, Quantitation Assay, Knockdown, Liquid Chromatography with Mass Spectroscopy, Control, Virus, Plaque Assay

Cell viability determined by MTT assay of Huh-7 cells treated with (a) non-targeting and ALKBH1-specific siRNA, and (b) control empty vector and ALKBH1 plasmid, at 24h and 48h post-transfection.

Journal: bioRxiv

Article Title: Dengue virus exploits the host tRNA epitranscriptome to promote viral replication

doi: 10.1101/2023.11.05.565734

Figure Lengend Snippet: Cell viability determined by MTT assay of Huh-7 cells treated with (a) non-targeting and ALKBH1-specific siRNA, and (b) control empty vector and ALKBH1 plasmid, at 24h and 48h post-transfection.

Techniques: MTT Assay, Control, Plasmid Preparation, Transfection

( a ) Illustration of ALKBH1 transcript with indicated siRNA-targeting sites. ( b ) Manipulation of ALKBH1 levels by siRNA knockdown, plasmid overexpression, and complementation using a siRNA-resistant ALKBH1 plasmid as assessed by immunoblotting. ( c ) LC-MS/MS profiling of modifications in f 5 Cm biogenesis of cells with indicated treatments versus cells treated with control siRNA and control plasmid. ( d ) Table of ALKBH1-dependent proteins obtained from proteomics analysis of treated cells from panel c . ( e ) Plot of p values derived from analysis of codon Z-scores of ALKBH1 negatively-regulated versus positively-regulated transcripts. ( f,g ) Z-score analysis (number of standard deviations above/below the mean) of leucine ( f ) and arginine ( g ) codons in ALKBH1 negatively-regulated (□) versus ALKBH1 positively-regulated (▪) transcripts. ( h ) Table of GO term and KEGG analysis of proteins deregulated by ALKBH1 depletion assessed by DAVID. Statistical significance was determined using Student’s t test, *p<0.05; **p<0.01.

Journal: bioRxiv

Article Title: Dengue virus exploits the host tRNA epitranscriptome to promote viral replication

doi: 10.1101/2023.11.05.565734

Figure Lengend Snippet: ( a ) Illustration of ALKBH1 transcript with indicated siRNA-targeting sites. ( b ) Manipulation of ALKBH1 levels by siRNA knockdown, plasmid overexpression, and complementation using a siRNA-resistant ALKBH1 plasmid as assessed by immunoblotting. ( c ) LC-MS/MS profiling of modifications in f 5 Cm biogenesis of cells with indicated treatments versus cells treated with control siRNA and control plasmid. ( d ) Table of ALKBH1-dependent proteins obtained from proteomics analysis of treated cells from panel c . ( e ) Plot of p values derived from analysis of codon Z-scores of ALKBH1 negatively-regulated versus positively-regulated transcripts. ( f,g ) Z-score analysis (number of standard deviations above/below the mean) of leucine ( f ) and arginine ( g ) codons in ALKBH1 negatively-regulated (□) versus ALKBH1 positively-regulated (▪) transcripts. ( h ) Table of GO term and KEGG analysis of proteins deregulated by ALKBH1 depletion assessed by DAVID. Statistical significance was determined using Student’s t test, *p<0.05; **p<0.01.

Techniques: Knockdown, Plasmid Preparation, Over Expression, Western Blot, Liquid Chromatography with Mass Spectroscopy, Control, Derivative Assay

(a) Densitometric quantitation of NS3 protein levels at indicated times after infection of Huh-7 cells with DENV strain EDEN2 infection. Huh-7 cells were pre-treated with control non-targeting siRNA (blue circles) and ALKBH1-specific siRNA (orange circles). (b) Differences in codon usage frequencies in DENV2 NGC strain versus human host genome. Leucine UUA codon (red arrow) is nearly equally represented in both human and DENV2 genomes, while other leucine codons (black arrows) are not shared equally.

Journal: bioRxiv

Article Title: Dengue virus exploits the host tRNA epitranscriptome to promote viral replication

doi: 10.1101/2023.11.05.565734

Figure Lengend Snippet: (a) Densitometric quantitation of NS3 protein levels at indicated times after infection of Huh-7 cells with DENV strain EDEN2 infection. Huh-7 cells were pre-treated with control non-targeting siRNA (blue circles) and ALKBH1-specific siRNA (orange circles). (b) Differences in codon usage frequencies in DENV2 NGC strain versus human host genome. Leucine UUA codon (red arrow) is nearly equally represented in both human and DENV2 genomes, while other leucine codons (black arrows) are not shared equally.

Techniques: Quantitation Assay, Infection, Control

In early stages of DENV infection, host cellular ALKBH1 levels decrease resulting in reduced f 5 Cm-modified ct-tRNA Leu(CAA) reduced decoding of non-cognate UUA codon. Transcripts lacking UUA are consequently preferentially translated and many of which have virus-associated functions, resulting in pro-viral translational remodeling of the host proteome to facilitate viral replication. As the virus replicates and translates its own mRNA, accumulating the MTase NS5 in the cell at later stages of infection, NS5 contributes to f 5 Cm-modification of tRNA, increasing UUA decoding and further cellular translational remodeling that may contribute to viral exocytosis.

Journal: bioRxiv

Article Title: Dengue virus exploits the host tRNA epitranscriptome to promote viral replication

doi: 10.1101/2023.11.05.565734

Figure Lengend Snippet: In early stages of DENV infection, host cellular ALKBH1 levels decrease resulting in reduced f 5 Cm-modified ct-tRNA Leu(CAA) reduced decoding of non-cognate UUA codon. Transcripts lacking UUA are consequently preferentially translated and many of which have virus-associated functions, resulting in pro-viral translational remodeling of the host proteome to facilitate viral replication. As the virus replicates and translates its own mRNA, accumulating the MTase NS5 in the cell at later stages of infection, NS5 contributes to f 5 Cm-modification of tRNA, increasing UUA decoding and further cellular translational remodeling that may contribute to viral exocytosis.

Techniques: Infection, Modification, Virus

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).

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.

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).

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.

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.

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.

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.

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

(A) Representative images of dot blot assay showing that the specific antibody against DNA N6-mA can detect the levels of N6-mA in mouse DRG neurons, the differentiated neuronal cell line CAD cells, and the Hela cells, in a dose-dependent manner. (B) Representative dot blot images showing that knocking down ALKBH1 in CAD cells led to increased level of N6-mA, whereas knocking down N6AMT1 alone had little effect. However, when ALKBH1 and N6AMT1 were knocked down together, the level of N6-mA was reversed back to the control condition. (C) Quantification of (B) (one-way ANOVA followed by Tukey’s multiple comparisons test, P = 0.0013, n = 4-5 independent experiments). (D) Representative dot blot images showing that treating the isolated DNA samples with DNase resulted in total loss of sample signals and associated N6-mA signals in either CAD or Hela cells. (E) Representative dot blot images showing that knocking down ALKBH1 in adult mouse DRG neurons led to increased level of N6-mA, whereas knocking down N6AMT1 alone had little effect. However, when ALKBH1 and N6AMT1 were knocked down together, the level of N6-mA was reversed back to the control condition. (F) Quantification of (B) (one-way ANOVA followed by Tukey’s multiple comparisons test, P = 0.002, n = 4 independent experiments). Data are represented as mean ± SEM. P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, compared to control if not designated.

Journal: bioRxiv

Article Title: N6-methyladenine DNA demethylase ALKBH1 regulates mammalian axon regeneration

doi: 10.1101/2020.08.19.258038

Figure Lengend Snippet: (A) Representative images of dot blot assay showing that the specific antibody against DNA N6-mA can detect the levels of N6-mA in mouse DRG neurons, the differentiated neuronal cell line CAD cells, and the Hela cells, in a dose-dependent manner. (B) Representative dot blot images showing that knocking down ALKBH1 in CAD cells led to increased level of N6-mA, whereas knocking down N6AMT1 alone had little effect. However, when ALKBH1 and N6AMT1 were knocked down together, the level of N6-mA was reversed back to the control condition. (C) Quantification of (B) (one-way ANOVA followed by Tukey’s multiple comparisons test, P = 0.0013, n = 4-5 independent experiments). (D) Representative dot blot images showing that treating the isolated DNA samples with DNase resulted in total loss of sample signals and associated N6-mA signals in either CAD or Hela cells. (E) Representative dot blot images showing that knocking down ALKBH1 in adult mouse DRG neurons led to increased level of N6-mA, whereas knocking down N6AMT1 alone had little effect. However, when ALKBH1 and N6AMT1 were knocked down together, the level of N6-mA was reversed back to the control condition. (F) Quantification of (B) (one-way ANOVA followed by Tukey’s multiple comparisons test, P = 0.002, n = 4 independent experiments). Data are represented as mean ± SEM. P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, compared to control if not designated.

Article Snippet: The siRNAs targeting mouse ALKBH1, N6AMT1 and non-targeting siRNA control were purchased from Dharmacon.

Techniques: Dot Blot, Control, Isolation

(A) Real-time PCR analysis showing significantly reduced mRNA level of ALKBH1 3 days after electroporation of siRNAs against ALKBH1 (siALKBH1) or together with siRNAs against N6AMT1 (siALK.+siN6A.). One sample t test, P = 0.0315 and 0.0092 for siALKBH1 and siALK.+siN6A., respectively. n = 3 independent experiments. (B) Real-time PCR analysis showing significantly reduced mRNA level of N6AMT1 3 days after electroporation of siRNAs against ALKBH1 and N6AMT1. One-sample t test, P < 0.0001, n = 3 independent experiments. (C) Top: time line of the culture and replate experiments. Bottom: representative images of cultured sensory neurons 24 hours post-replating after knocking down ALKBH1, N6AMT1, or together with siRNAs (siALKBH1, siN6AMT1, or siALK.+siN6A.). Scale bar, 500 μm. (D) Quantification of average lengths of the longest neurites (one-way ANOVA followed by Tukey’s multiple comparisons test, P < 0.0001, n = 7-10 independent experiments). (E) Quantification of percentage of neurons with axons (one-way ANOVA followed by Tukey’s multiple comparisons test, P = 0.0827, n = 4 independent experiments). Data are represented as mean ± SEM. P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, compared to control if not designated.

Journal: bioRxiv

Article Title: N6-methyladenine DNA demethylase ALKBH1 regulates mammalian axon regeneration

doi: 10.1101/2020.08.19.258038

Figure Lengend Snippet: (A) Real-time PCR analysis showing significantly reduced mRNA level of ALKBH1 3 days after electroporation of siRNAs against ALKBH1 (siALKBH1) or together with siRNAs against N6AMT1 (siALK.+siN6A.). One sample t test, P = 0.0315 and 0.0092 for siALKBH1 and siALK.+siN6A., respectively. n = 3 independent experiments. (B) Real-time PCR analysis showing significantly reduced mRNA level of N6AMT1 3 days after electroporation of siRNAs against ALKBH1 and N6AMT1. One-sample t test, P < 0.0001, n = 3 independent experiments. (C) Top: time line of the culture and replate experiments. Bottom: representative images of cultured sensory neurons 24 hours post-replating after knocking down ALKBH1, N6AMT1, or together with siRNAs (siALKBH1, siN6AMT1, or siALK.+siN6A.). Scale bar, 500 μm. (D) Quantification of average lengths of the longest neurites (one-way ANOVA followed by Tukey’s multiple comparisons test, P < 0.0001, n = 7-10 independent experiments). (E) Quantification of percentage of neurons with axons (one-way ANOVA followed by Tukey’s multiple comparisons test, P = 0.0827, n = 4 independent experiments). Data are represented as mean ± SEM. P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, compared to control if not designated.

Article Snippet: The siRNAs targeting mouse ALKBH1, N6AMT1 and non-targeting siRNA control were purchased from Dharmacon.

Techniques: Real-time Polymerase Chain Reaction, Electroporation, Cell Culture, Control

(A) Real-time PCR analysis showing significantly elevated mRNA level of ALKBH1 in sensory neurons 1-day post-nerve injury (one sample t test, P = 0.0014, n = 4 independent experiments). (B) Real-time PCR analysis showing significantly reduced mRNA level of ALKBH1 in vivo after 3 days after electroporation (one sample t test, P = 0.0076, n = 4 independent experiments). (C) Top: timeline of the experiment. Bottom: representative images showing that knocking down ALKBH1 had no effect on slower sensory axon regeneration 2 days post-nerve injury. The right column shows enlarged images of areas in the dashed yellow boxes. The red line indicates the crush sites and the read arrows label regenerating axon tips. Scale bar, 1 mm for the left panel and 0.5 mm for the right panel. (D) Quantification of average length of regenerating sensory axons showing no significant difference between the control group and the ALKBH1 knockdown group 2 days post-nerve injury (unpaired student t test, P = 0.6387, n = 7 mice in each condition). (E) Cumulative distribution curves showing similar axon regeneration between the control group and the ALKBH1 knockdown group 2 days post-nerve injury. (F) Top: timeline of the experiment. Bottom: representative images showing that knocking down N6AMT1 had no effect on slower sensory axon regeneration 2 days post-nerve injury. The right column shows enlarged images of areas in the dashed yellow boxes. The red line indicates the crush sites and the read arrows label regenerating axon tips. Scale bar, 1 mm for the left panel and 0.5 mm for the right panel. (G) Quantification of average length of regenerating sensory axons showing no significant difference between the control group and the N6AMT1 knockdown group 2 days post-nerve injury (unpaired student t test, P = 0.2715, n = 7 and 10 mice in control and siN6AMT1 group, respectively). (H) Cumulative distribution curves showing similar axon regeneration between the control group and the N6AMT1 knockdown group 2 days post-nerve injury. Data are represented as mean ± SEM. P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, compared to control if not designated.

Journal: bioRxiv

Article Title: N6-methyladenine DNA demethylase ALKBH1 regulates mammalian axon regeneration

doi: 10.1101/2020.08.19.258038

Figure Lengend Snippet: (A) Real-time PCR analysis showing significantly elevated mRNA level of ALKBH1 in sensory neurons 1-day post-nerve injury (one sample t test, P = 0.0014, n = 4 independent experiments). (B) Real-time PCR analysis showing significantly reduced mRNA level of ALKBH1 in vivo after 3 days after electroporation (one sample t test, P = 0.0076, n = 4 independent experiments). (C) Top: timeline of the experiment. Bottom: representative images showing that knocking down ALKBH1 had no effect on slower sensory axon regeneration 2 days post-nerve injury. The right column shows enlarged images of areas in the dashed yellow boxes. The red line indicates the crush sites and the read arrows label regenerating axon tips. Scale bar, 1 mm for the left panel and 0.5 mm for the right panel. (D) Quantification of average length of regenerating sensory axons showing no significant difference between the control group and the ALKBH1 knockdown group 2 days post-nerve injury (unpaired student t test, P = 0.6387, n = 7 mice in each condition). (E) Cumulative distribution curves showing similar axon regeneration between the control group and the ALKBH1 knockdown group 2 days post-nerve injury. (F) Top: timeline of the experiment. Bottom: representative images showing that knocking down N6AMT1 had no effect on slower sensory axon regeneration 2 days post-nerve injury. The right column shows enlarged images of areas in the dashed yellow boxes. The red line indicates the crush sites and the read arrows label regenerating axon tips. Scale bar, 1 mm for the left panel and 0.5 mm for the right panel. (G) Quantification of average length of regenerating sensory axons showing no significant difference between the control group and the N6AMT1 knockdown group 2 days post-nerve injury (unpaired student t test, P = 0.2715, n = 7 and 10 mice in control and siN6AMT1 group, respectively). (H) Cumulative distribution curves showing similar axon regeneration between the control group and the N6AMT1 knockdown group 2 days post-nerve injury. Data are represented as mean ± SEM. P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, compared to control if not designated.

Article Snippet: The siRNAs targeting mouse ALKBH1, N6AMT1 and non-targeting siRNA control were purchased from Dharmacon.

Techniques: Real-time Polymerase Chain Reaction, In Vivo, Electroporation, Control, Knockdown

(A) Top: timeline of the experiment. Bottom: representative images showing that knocking down ALKBH1 significantly impaired fast sensory axon regeneration 3 days post-nerve injury. The right column shows enlarged images of areas in the dashed yellow boxes. The red line indicates the crush sites and the read arrows label regenerating axon tips. Scale bar, 1 mm for the left panel and 0.5 mm for the right panel. (B) Quantification of average length of regenerating axons showing that knocking down ALKBH1 significantly impaired sensory axon regeneration 3 days post-nerve injury (unpaired student t test, P < 0.0001, n = 8 mice in each condition). (C) Cumulative distribution curves showing that ALKBH1 knockdown resulted in significant reduced sensory axon regeneration 3 days post-nerve injury. (D) Top: timeline of the experiment. Bottom: representative images showing that knocking down ALKBH1 significantly impaired fast sensory axon regeneration 4 days post-nerve injury. The right column shows enlarged images of areas in the dashed yellow boxes. The red line indicates the crush sites and the read arrows label regenerating axon tips. Scale bar, 1 mm for the left panel and 0.5 mm for the right panel. (E) Quantification of average length of regenerating axons showing that knocking down ALKBH1 significantly impaired sensory axon regeneration 4 days post-nerve injury (unpaired student t test, P = 0.018, n = 4 mice in each condition). (F) Cumulative distribution curves showing that ALKBH1 knockdown resulted in significant reduced sensory axon regeneration 4 days post-nerve injury. Data are represented as mean ± SEM. P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, compared to control if not designated.

Journal: bioRxiv

Article Title: N6-methyladenine DNA demethylase ALKBH1 regulates mammalian axon regeneration

doi: 10.1101/2020.08.19.258038

Figure Lengend Snippet: (A) Top: timeline of the experiment. Bottom: representative images showing that knocking down ALKBH1 significantly impaired fast sensory axon regeneration 3 days post-nerve injury. The right column shows enlarged images of areas in the dashed yellow boxes. The red line indicates the crush sites and the read arrows label regenerating axon tips. Scale bar, 1 mm for the left panel and 0.5 mm for the right panel. (B) Quantification of average length of regenerating axons showing that knocking down ALKBH1 significantly impaired sensory axon regeneration 3 days post-nerve injury (unpaired student t test, P < 0.0001, n = 8 mice in each condition). (C) Cumulative distribution curves showing that ALKBH1 knockdown resulted in significant reduced sensory axon regeneration 3 days post-nerve injury. (D) Top: timeline of the experiment. Bottom: representative images showing that knocking down ALKBH1 significantly impaired fast sensory axon regeneration 4 days post-nerve injury. The right column shows enlarged images of areas in the dashed yellow boxes. The red line indicates the crush sites and the read arrows label regenerating axon tips. Scale bar, 1 mm for the left panel and 0.5 mm for the right panel. (E) Quantification of average length of regenerating axons showing that knocking down ALKBH1 significantly impaired sensory axon regeneration 4 days post-nerve injury (unpaired student t test, P = 0.018, n = 4 mice in each condition). (F) Cumulative distribution curves showing that ALKBH1 knockdown resulted in significant reduced sensory axon regeneration 4 days post-nerve injury. Data are represented as mean ± SEM. P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, compared to control if not designated.

Article Snippet: The siRNAs targeting mouse ALKBH1, N6AMT1 and non-targeting siRNA control were purchased from Dharmacon.

Techniques: Knockdown, Control

(A) Top: timeline of the experiment. Bottom: representative images showing that knocking down N6AMT1 reversed sensory axon regeneration impaired by down regulation of ALKBH1 3 days post-nerve injury. The right column shows enlarged images of areas in the dashed yellow boxes. The red line indicates the crush sites and the read arrows label regenerating axon tips. Scale bar, 1 mm for the left panel and 0.5 mm for the right panel. (B) Quantification of average length of regenerating axons under conditions shown in (A). One-way ANOVA followed by Tukey’s multiple comparisons test, P < 0.0001, n = 7-9 mice in each condition. (C) Cumulative distribution curves showing that knocking down N6AMT1 rescued axon regeneration impaired by ALKBH1 knockdown. Data are represented as mean ± SEM. P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, compared to control if not designated.

Journal: bioRxiv

Article Title: N6-methyladenine DNA demethylase ALKBH1 regulates mammalian axon regeneration

doi: 10.1101/2020.08.19.258038

Figure Lengend Snippet: (A) Top: timeline of the experiment. Bottom: representative images showing that knocking down N6AMT1 reversed sensory axon regeneration impaired by down regulation of ALKBH1 3 days post-nerve injury. The right column shows enlarged images of areas in the dashed yellow boxes. The red line indicates the crush sites and the read arrows label regenerating axon tips. Scale bar, 1 mm for the left panel and 0.5 mm for the right panel. (B) Quantification of average length of regenerating axons under conditions shown in (A). One-way ANOVA followed by Tukey’s multiple comparisons test, P < 0.0001, n = 7-9 mice in each condition. (C) Cumulative distribution curves showing that knocking down N6AMT1 rescued axon regeneration impaired by ALKBH1 knockdown. Data are represented as mean ± SEM. P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, compared to control if not designated.

Article Snippet: The siRNAs targeting mouse ALKBH1, N6AMT1 and non-targeting siRNA control were purchased from Dharmacon.

Techniques: Knockdown, Control

(A) Real-time PCR analyses of changed transcription of 18 genes in sensory neurons after knocking down ALKBH1. The 5 significantly down regulated genes were underlined in red. One sample t test, P = 0.0096, 0.0118, and 0.0202 for Efna1, Id1, and Nrn1 , respectively. P < 0.0001 for ATG9B or C1QL4 . n = 3 independent experiments. (B) Real-time PCR analysis of ATG9B mRNA levels after knocking down ALKBH1 or double knocking down ALKBH1 and N6AMT1. One-way ANOVA followed by Tukey’s multiple comparisons test, P < 0.0001, n = 3 independent experiments. (C) Real-time PCR analysis of C1QL4 mRNA levels after knocking down ALKBH1 or double knocking down ALKBH1 and N6AMT1. One-way ANOVA followed by Tukey’s multiple comparisons test, P < 0.0001, n = 3 independent experiments. (D) Real-time PCR analysis of Id1 mRNA levels after knocking down ALKBH1 or double knocking down ALKBH1 and N6AMT1. One-way ANOVA followed by Tukey’s multiple comparisons test, P = 0.0001, n = 3 independent experiments. (E) Real-time PCR analysis of Nrn1 mRNA levels after knocking down ALKBH1 or double knocking down ALKBH1 and N6AMT1. One-way ANOVA followed by Tukey’s multiple comparisons test, P = 0.0368, n = 3 independent experiments. Data are represented as mean ± SEM. P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, compared to control if not designated.

Journal: bioRxiv

Article Title: N6-methyladenine DNA demethylase ALKBH1 regulates mammalian axon regeneration

doi: 10.1101/2020.08.19.258038

Figure Lengend Snippet: (A) Real-time PCR analyses of changed transcription of 18 genes in sensory neurons after knocking down ALKBH1. The 5 significantly down regulated genes were underlined in red. One sample t test, P = 0.0096, 0.0118, and 0.0202 for Efna1, Id1, and Nrn1 , respectively. P < 0.0001 for ATG9B or C1QL4 . n = 3 independent experiments. (B) Real-time PCR analysis of ATG9B mRNA levels after knocking down ALKBH1 or double knocking down ALKBH1 and N6AMT1. One-way ANOVA followed by Tukey’s multiple comparisons test, P < 0.0001, n = 3 independent experiments. (C) Real-time PCR analysis of C1QL4 mRNA levels after knocking down ALKBH1 or double knocking down ALKBH1 and N6AMT1. One-way ANOVA followed by Tukey’s multiple comparisons test, P < 0.0001, n = 3 independent experiments. (D) Real-time PCR analysis of Id1 mRNA levels after knocking down ALKBH1 or double knocking down ALKBH1 and N6AMT1. One-way ANOVA followed by Tukey’s multiple comparisons test, P = 0.0001, n = 3 independent experiments. (E) Real-time PCR analysis of Nrn1 mRNA levels after knocking down ALKBH1 or double knocking down ALKBH1 and N6AMT1. One-way ANOVA followed by Tukey’s multiple comparisons test, P = 0.0368, n = 3 independent experiments. Data are represented as mean ± SEM. P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, compared to control if not designated.

Article Snippet: The siRNAs targeting mouse ALKBH1, N6AMT1 and non-targeting siRNA control were purchased from Dharmacon.

Techniques: Real-time Polymerase Chain Reaction, Control

Impact of Alkbh1 knockdown on cell fate and tRNA cleavage after stress. A: Annexin V FACS analysis (left graph) and SYBR gold staining following Alkbh1 KD and arsenite stress (400 µM for 4 hours). Alkbh1 KD reduced the levels of apoptotic and necrotic cells and rescued tRNA from cleavage. B: Annexin V FACS analysis and SYBR gold staining following Alkbh1 KD and antimycin stress (150 µg/ml for 4 hours). Alkbh1 KD had no impact on cell fate or tRNA cleavage following antimycin stress. (Note: FACS for As and antimycin stresses were performed simultaneously but the results presented in 2 graphs for easier comprehension and data presentation. FACS data were presented as ratio to unstressed (control) cells). C: Annexin V FACS analysis and SYBR gold staining following Alkbh1 KD and OGD-R stress. Alkbh1 KD also had no impact on cell death ratio following OGD-R and no apparent impact on tRNA cleavage. Asterisk: p < 0.05, N.S: not significant statistically. SYBR gold staining was performed with 500ng total RNA per lane.

Journal: RNA Biology

Article Title: The stress specific impact of ALKBH1 on tRNA cleavage and tiRNA generation

doi: 10.1080/15476286.2020.1779492

Figure Lengend Snippet: Impact of Alkbh1 knockdown on cell fate and tRNA cleavage after stress. A: Annexin V FACS analysis (left graph) and SYBR gold staining following Alkbh1 KD and arsenite stress (400 µM for 4 hours). Alkbh1 KD reduced the levels of apoptotic and necrotic cells and rescued tRNA from cleavage. B: Annexin V FACS analysis and SYBR gold staining following Alkbh1 KD and antimycin stress (150 µg/ml for 4 hours). Alkbh1 KD had no impact on cell fate or tRNA cleavage following antimycin stress. (Note: FACS for As and antimycin stresses were performed simultaneously but the results presented in 2 graphs for easier comprehension and data presentation. FACS data were presented as ratio to unstressed (control) cells). C: Annexin V FACS analysis and SYBR gold staining following Alkbh1 KD and OGD-R stress. Alkbh1 KD also had no impact on cell death ratio following OGD-R and no apparent impact on tRNA cleavage. Asterisk: p < 0.05, N.S: not significant statistically. SYBR gold staining was performed with 500ng total RNA per lane.

Article Snippet: Rat Alkbh1 siRNA was purchased from Thermo Fischer (Silencer select siRNA, Cat# 4390771, siRNA ID: s169728).

Techniques: Staining

Review

Structured Review

Images

1) Product Images from "ALKBH1-demethylated DNA N 6 -methyladenine modification triggers vascular calcification via osteogenic reprogramming in chronic kidney disease"

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