Journal: The Journal of Biological Chemistry

Article Title: DNA demethylase ALKBH1 promotes adipogenic differentiation via regulation of HIF-1 signaling

doi: 10.1016/j.jbc.2021.101499

Figure Lengend Snippet: ALKBH1 is upregulated during adipogenic differentiation. A, immunofluorescence staining of ALKBH1 and Nile red staining in the iWAT of C57BL/6J mice. ALKBH1 is expressed widely in adipose. Scale bar: 30 μm. B, Western blot analysis of ALKBH1 and adipogenic markers in hMSCs cells during adipogenic differentiation. C, qRT-PCR analysis of mRNA level of ALKBH1 in hMSCs after adipogenic induction (n = 3). D, Western blot analysis of ALKBH1, CEBPA, and FABP4 in 3T3-L1 cells during adipogenic differentiation. E, qRT-PCR analysis of the expression of Alkbh1 in 3T3-L1 preadipocytes during adipogenic differentiation (n = 3). The p values were calculated by two-tailed Student's t test. Scatter plots show individual data points ± SD.

Article Snippet: siRNAs for human ALKBH1 and control were purchased from Santa Cruz.

Techniques: Immunofluorescence, Staining, Western Blot, Quantitative RT-PCR, Expressing, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: DNA demethylase ALKBH1 promotes adipogenic differentiation via regulation of HIF-1 signaling

doi: 10.1016/j.jbc.2021.101499

Figure Lengend Snippet: Depletion of ALKBH1 inhibits adipogenic differentiation in vitro. A, Western blot analysis confirms successful knockdown of ALKBH1 in hMSCs (n = 3). B, CCK8 assay shows the viability of hMSCs is not affected after ALKBH1 depletion (n = 3). The absorbance was analyzed at 450 nm at 48, 72, and 96 h. C, representative images and quantitative analysis of oil red O staining. hMSCs were transfected with siRNA and subjected to adipogenic induction for 14 days. Lipid droplets were dissolved by isopropanol and quantified at 500 nm (n = 3). Scale bar: 25 μm. D, qRT-PCR analysis of the mRNA expression of adipogenic related genes CEBPA , PPARG , PLIN1, ADIPOQ after 2- and 5-day adipogenic differentiation (n = 3). E, Western blot shows successful knockdown of ALKBH1 in 3T3-L1 cells. F, CCK8 assay of viability of 3T3-L1 cells after knockdown of Alkbh1 . G, representative images and quantitation of oil red O staining in 3T3-L1 cells after induction of 14 days (n = 3). Scale bar: 25 μm. H, qRT-PCR analysis of Cebpα, Pparγ, Plin1, Adipoq in Alkbh1 -knockdown 3T3-L1 cells (n = 3). The p values were calculated by two-tailed Student's t test. Scatter plots show individual data points ± SD.

Article Snippet: siRNAs for human ALKBH1 and control were purchased from Santa Cruz.

Techniques: In Vitro, Western Blot, CCK-8 Assay, Staining, Transfection, Quantitative RT-PCR, Expressing, Quantitation Assay, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: DNA demethylase ALKBH1 promotes adipogenic differentiation via regulation of HIF-1 signaling

doi: 10.1016/j.jbc.2021.101499

Figure Lengend Snippet: Overexpression of ALKBH1 promotes adipogenesis. A, Western blot confirms the successful overexpression of ALKBH1 in hMSCs. B, quantitative analysis of oil red O staining in hMSCs after 14 days of adipogenic differentiation (n = 3). Scale bar: 25 μm. C, qRT-PCR analysis of adipogenic related genes CEBPA , PPARG , PLIN1, ADIPOQ in ALKBH1 -overexpressed hMSCs (n = 3). D, Western blot shows overexpression of ALKBH1 in 3T3-L1 cells. E, representative images and the OD values of oil red O staining in 3T3-L1 cells (n = 3). Scale bar: 25 μm. F, qRT-PCR analysis of Cebpα , Pparγ , Plin1, Adipoq in ALKBH1 -overexpressed 3T3-L1 cells (n = 3). G, macroscopic identification and quantitative analysis of ectopic fats after 6 weeks of sternal subcutaneous injection. Nude mice were bilateral transplanted with Vector and Lv- ALKBH1 3T3-L1 cells (n = 6). Scale bar: 5 mm. H, hematoxylin and eosin staining ( top ) and GFP staining ( bottom ) of fat pads isolated from nude mice. Scale bar: 25 μm. The p values were calculated by two-tailed Student's t test. Scatter plots show individual data points ± SD.

Article Snippet: siRNAs for human ALKBH1 and control were purchased from Santa Cruz.

Techniques: Over Expression, Western Blot, Staining, Quantitative RT-PCR, Injection, Plasmid Preparation, Isolation, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: DNA demethylase ALKBH1 promotes adipogenic differentiation via regulation of HIF-1 signaling

doi: 10.1016/j.jbc.2021.101499

Figure Lengend Snippet: ALKBH1 regulates HIF-1 pathway. A, Gene Ontology enrichment interprets the biological process in ALKBH1 -knockdown hMSCs. Knockdown of ALKBH1 influenced the synthesis and transport of lipid. B, gene set enrichment analysis (GSEA) shows decreased expression of adipogenesis genes in ALKBH1 -knockdown hMSCs. C, GSEA reveals that depletion of ALKBH1 in hMSCs impairs hypoxia pathway. D, heatmap of representative genes involved in HIF-1 signaling. E, Western blots of hypoxia-related proteins in Alkbh1 -knocked 3T3-L1 cells in 20% oxygen conditions. F, qRT-PCR analysis of Hif-1α, Ca9, Hk2, Vegfα, Glut1, and Ldha in Alkbh1 -deficient 3T3-L1 lines in 20% oxygen conditions (n = 3). G, Western blots and ( H ) qRT-PCR analysis confirm the activation of hypoxia axis in ALKBH1 -overexpressed cells under normoxia (n = 3). I, qRT-PCR analysis of the mRNA levels of HIF-1 pathway. HIF-1 pathway activator ML228 upregulated related genes expression in control group but partially failed in Alkbh1 -knockdown group (n = 3). The p values were calculated by two-tailed Student's t test ( F and H ) and one-way ANOVA with Tukey's post hoc test ( I ). Scatter plots show individual data points ± SD.

Article Snippet: siRNAs for human ALKBH1 and control were purchased from Santa Cruz.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Activation Assay, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: DNA demethylase ALKBH1 promotes adipogenic differentiation via regulation of HIF-1 signaling

doi: 10.1016/j.jbc.2021.101499

Figure Lengend Snippet: ALKBH1 demethylates DNA 6 mA of H IF -1α. A, levels of the DNA 6mA modification were assessed via DNA dot blot in 3T3-L1 cells using a 6mA-specific antibody. Methylene blue detected DNA loading. ALKBH1 functions as a DNA demethylase in 3T3-L1 cells. si-Ctrl : control-siRNA 3T3-L1 cells, si-Alkbh1 : Alkbh1 deficient 3T3-L1 cells, Lv- ALKBH1 : ALKBH1 -overexpressed 3T3-L1 cells, Vector: Empty vector in 3T3-L1 cells. B, enrichment of N6-mA peak on Hif-1α gene locus in Alkbh1 knockout embryonic stem cells. Data are from the published datasets GSE71866. C, ChIP-PCR analysis of immunoprecipitations of ALKBH1 associated Hif-1α fragments. D, representative images of Oil red O staining in 3T3-L1 cells and quantitation analysis (n = 3). Scale bar: 25 μm. E, Western blot analysis of HIF-1α, CEBPA and FABP4. Overexpression of HIF-1α partially rescued the decrease of adipogenic markers. F, qRT-PCR shows the mRNA level of Cebpα , Pparγ , Plin1, and Adipoq in si- Ctrl +Vector, si- Alkbh1 +Vector, and si- Alkbh1 +Lv- HIF1α group at 5 days of differentiation (n = 3). The p values were calculated by two-tailed Student' t test ( C ) and one-way ANOVA with Tukey's post hoc test ( D and F ). Scatter plots show individual data points ± SD.

Article Snippet: siRNAs for human ALKBH1 and control were purchased from Santa Cruz.

Techniques: Modification, Dot Blot, Plasmid Preparation, Knock-Out, Staining, Quantitation Assay, Western Blot, Over Expression, Quantitative RT-PCR, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: DNA demethylase ALKBH1 promotes adipogenic differentiation via regulation of HIF-1 signaling

doi: 10.1016/j.jbc.2021.101499

Figure Lengend Snippet: Simultaneous overexpression of HIF-1α and GYS1 restores the adipogenic differentiation of Alkbh1 -deficient cells. A, DNA N6-mA peaks on Gys1 gene locus in Alkbh1 knockout embryonic stem cells. Data are from the published datasets GSE71866. B, ChIP-PCR analysis of immunoprecipitations of ALKBH1 associated Gys1 fragments (n = 3). C, Western blots of GYS1 in normoxia, indicating that ALKBH1 regulates the expression of GYS1. D, representative images and quantification of oil red O staining in si- Ctrl +Vector, si- Alkbh1 +Vector, si- Alkbh1 +Lv- HIF1α , si- Alkbh1 +Lv- GYS1 group, and si- Alkbh1 +Lv- HIF1α + GYS1 group (n = 3). Scale bar: 25 μm. E, Western blot detects the protein level of HIF-1α, GYS1 and adipogenic-related markers (n = 3). Simultaneous overexpression of HIF-1α and GYS1 restored adipogenic differentiation of Alkbh1 -deficient cells. F, qRT-PCR analysis of the expression of Cebpα , Pparγ , Plin1, Adipoq after 5-day adipogenic differentiation (n = 3). The p values were calculated by two-tailed Student's t test ( B ) and one-way ANOVA with Tukey's post hoc test ( D and F ). Scatter plots show individual data points ± SD.

Article Snippet: siRNAs for human ALKBH1 and control were purchased from Santa Cruz.

Techniques: Over Expression, Knock-Out, Western Blot, Expressing, Staining, Plasmid Preparation, Quantitative RT-PCR, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: N 6 -Hydroxymethyladenine: a hydroxylation derivative of N 6 -methyladenine in genomic DNA of mammals

doi: 10.1093/nar/gky1218

Figure Lengend Snippet: Schematic illustration of the formation of hm 6 dA in DNA from the hydroxylation of m 6 dA by the Fe 2+ - and 2-oxoglutarate-dependent ALKBH1 protein.

Article Snippet: RNA knockdown of ALKBH1 was performed using siRNA (Takara Biotechnology) against human ALKBH1 mRNA.

Techniques:

Journal: Nucleic Acids Research

Article Title: N 6 -Hydroxymethyladenine: a hydroxylation derivative of N 6 -methyladenine in genomic DNA of mammals

doi: 10.1093/nar/gky1218

Figure Lengend Snippet: ALKBH1 oxidizes m 6 dA to form hm 6 dA in vitro . ( A ) Amounts of detected hm 6 dA in the in-vitro reaction under different conditions. ( B ) Amounts of detected m 6 dA in the in-vitro reaction under different conditions. ( C ) Time-dependent formation of hm 6 dA in single-stranded DNA. ( D ) Amounts of hm 6 dA and m 6 dA with the increased amount of ALKBH1 protein. WT, wild-type ALKBH1 protein; D233A, mutant ALKBH1 protein (D233A); 2-KG, 2-oxoglutarate. ND, not detected. ** P < 0.01 ( n = 3). NS, not significant. The ANOVA test was performed to evaluate the differences of m 6 dA modification between multiple conditions of in-vitro biochemical assay.

Article Snippet: RNA knockdown of ALKBH1 was performed using siRNA (Takara Biotechnology) against human ALKBH1 mRNA.

Techniques: In Vitro, Mutagenesis, Modification

Journal: Nucleic Acids Research

Article Title: N 6 -Hydroxymethyladenine: a hydroxylation derivative of N 6 -methyladenine in genomic DNA of mammals

doi: 10.1093/nar/gky1218

Figure Lengend Snippet: Overexpression and knockdown of ALKBH1 induced the contents change of m 6 dA and hm 6 dA. ( A ) Measured m 6 dA in HEK293T cells upon overexpression and knockdown of ALKBH1 . ( B ) Measured hm 6 dA in HEK293T cells upon overexpression and knockdown of ALKBH1 . ** P < 0.01 ( n = 3). Two-side unpaired t -test was performed.

Article Snippet: RNA knockdown of ALKBH1 was performed using siRNA (Takara Biotechnology) against human ALKBH1 mRNA.

Techniques: Over Expression

Journal: Nucleic Acids Research

Article Title: N 6 -Hydroxymethyladenine: a hydroxylation derivative of N 6 -methyladenine in genomic DNA of mammals

doi: 10.1093/nar/gky1218

Figure Lengend Snippet: Quantification and statistical analysis of DNA modifications and the relative mRNA level of ALKBH1 in human lung carcinoma tissues and tumor-adjacent normal tissues. Quantification and statistical analysis of m 6 dA ( A ), hm 6 dA ( B ), m 5 dC ( C ) and hm 5 dC ( D ) by LC–MS/MS. ( E ) The relative mRNA level of ALKBH1. GAPDH was used as endogenous control and triplicate measurements were carried out. A total of 20 tissues from 10 lung cancer patients were analysed. Two-side paired t -test was performed.

Article Snippet: RNA knockdown of ALKBH1 was performed using siRNA (Takara Biotechnology) against human ALKBH1 mRNA.

Techniques: Liquid Chromatography with Mass Spectroscopy

Journal: Nucleic Acids Research

Article Title: N 6 -Hydroxymethyladenine: a hydroxylation derivative of N 6 -methyladenine in genomic DNA of mammals

doi: 10.1093/nar/gky1218

Figure Lengend Snippet: Schematic illustration of the formation of hm 6 dA in DNA from the hydroxylation of m 6 dA by the Fe 2+ - and 2-oxoglutarate-dependent ALKBH1 protein.

Article Snippet: To express the recombinant human ALKBH1protein in E. coli , the pGEX-ALKBH1 plasmid was constructed by inserting the full length coding sequence of human ALKBH1 into the vector of pGEX-4T1, which contains the glutathione S-transferase (GST) tag and Eco R I/ Xho I cloning sites (GE Healthcare, USA).

Techniques:

Journal: Nucleic Acids Research

Article Title: N 6 -Hydroxymethyladenine: a hydroxylation derivative of N 6 -methyladenine in genomic DNA of mammals

doi: 10.1093/nar/gky1218

Figure Lengend Snippet: ALKBH1 oxidizes m 6 dA to form hm 6 dA in vitro . ( A ) Amounts of detected hm 6 dA in the in-vitro reaction under different conditions. ( B ) Amounts of detected m 6 dA in the in-vitro reaction under different conditions. ( C ) Time-dependent formation of hm 6 dA in single-stranded DNA. ( D ) Amounts of hm 6 dA and m 6 dA with the increased amount of ALKBH1 protein. WT, wild-type ALKBH1 protein; D233A, mutant ALKBH1 protein (D233A); 2-KG, 2-oxoglutarate. ND, not detected. ** P < 0.01 ( n = 3). NS, not significant. The ANOVA test was performed to evaluate the differences of m 6 dA modification between multiple conditions of in-vitro biochemical assay.

Article Snippet: To express the recombinant human ALKBH1protein in E. coli , the pGEX-ALKBH1 plasmid was constructed by inserting the full length coding sequence of human ALKBH1 into the vector of pGEX-4T1, which contains the glutathione S-transferase (GST) tag and Eco R I/ Xho I cloning sites (GE Healthcare, USA).

Techniques: In Vitro, Mutagenesis, Modification

Journal: Nucleic Acids Research

Article Title: N 6 -Hydroxymethyladenine: a hydroxylation derivative of N 6 -methyladenine in genomic DNA of mammals

doi: 10.1093/nar/gky1218

Figure Lengend Snippet: Overexpression and knockdown of ALKBH1 induced the contents change of m 6 dA and hm 6 dA. ( A ) Measured m 6 dA in HEK293T cells upon overexpression and knockdown of ALKBH1 . ( B ) Measured hm 6 dA in HEK293T cells upon overexpression and knockdown of ALKBH1 . ** P < 0.01 ( n = 3). Two-side unpaired t -test was performed.

Article Snippet: To express the recombinant human ALKBH1protein in E. coli , the pGEX-ALKBH1 plasmid was constructed by inserting the full length coding sequence of human ALKBH1 into the vector of pGEX-4T1, which contains the glutathione S-transferase (GST) tag and Eco R I/ Xho I cloning sites (GE Healthcare, USA).

Techniques: Over Expression

Journal: Nucleic Acids Research

Article Title: N 6 -Hydroxymethyladenine: a hydroxylation derivative of N 6 -methyladenine in genomic DNA of mammals

doi: 10.1093/nar/gky1218

Figure Lengend Snippet: Quantification and statistical analysis of DNA modifications and the relative mRNA level of ALKBH1 in human lung carcinoma tissues and tumor-adjacent normal tissues. Quantification and statistical analysis of m 6 dA ( A ), hm 6 dA ( B ), m 5 dC ( C ) and hm 5 dC ( D ) by LC–MS/MS. ( E ) The relative mRNA level of ALKBH1. GAPDH was used as endogenous control and triplicate measurements were carried out. A total of 20 tissues from 10 lung cancer patients were analysed. Two-side paired t -test was performed.

Article Snippet: To express the recombinant human ALKBH1protein in E. coli , the pGEX-ALKBH1 plasmid was constructed by inserting the full length coding sequence of human ALKBH1 into the vector of pGEX-4T1, which contains the glutathione S-transferase (GST) tag and Eco R I/ Xho I cloning sites (GE Healthcare, USA).

Techniques: Liquid Chromatography with Mass Spectroscopy

Journal: Nucleic Acids Research

Article Title: ALKBH1 is an RNA dioxygenase responsible for cytoplasmic and mitochondrial tRNA modifications

doi: 10.1093/nar/gkx354

Figure Lengend Snippet: In vitro reconstitution of f 5 C34 formation ( A ) Mass chromatograms of the anticodon-containing fragments of mt-tRNA Met and ASL to detect f 5 C34 (top), hm 5 C34 (middle) and m 5 C34 (bottom) after in vitro reconstitution of f 5 C34 with ALKBH1 in the presence or absence of 2-OG and Fe 2+ . ( B ) CID spectra of the hm 5 C34 (left)- and f 5 C34 (right)-containing fragments reconstituted in vitro . The precursor ions are m/z 1163.48 and 1163.15 for hm 5 C34 and f 5 C34, respectively. Product ions are assigned on the corresponding sequences. ( C ) In vitro f 5 C34 formation from C34 via consecutive reactions mediated by NSUN3 and ALKBH1. Time-dependent alteration of modification status in mt-tRNA Met . Fractions of the RNase T 1 -digested fragments bearing C34 (green), m 5 C34 (pale green), hm 5 C34 (pale orange) and f 5 C34 (orange) at each time point were calculated based on peak heights of mass chromatograms. Because hm 5 C has a molecular mass 2Da larger than that of f 5 C, the frequency of hm 5 C was calculated by subtracting the theoretical value of the second isotopic peak (+2) of the f 5 C-containing fragment. ( D ) Methylation (gray bars) and oxidation (orange bars) efficiencies of mutant mt-tRNAs Met normalized against those of mt-tRNA Met . Positions of pathogenic mutations in mt-tRNA Met are shown in Figure . Methylation data were obtained from our previous study . Each mt-tRNA Met mutant containing m 5 C34 was incubated with ALKBH1 in the presence of 2-OG and Fe 2+ , followed by RNase T 1 digestion and subjected to RNA-MS. Reaction efficiency was calculated by summing the peak heights of the anticodon-containing fragments bearing hm 5 C34 and f 5 C34, normalized against the total peak heights of the three fragments (m 5 C34, hm 5 C34 and f 5 C34). Frequency of hm 5 C was calculated by subtracting the theoretical value of the second isotopic peak (+2) of the f 5 C-containing fragment. Data are presented as means ± SD of three independent reactions. * P < 0.001, Student's t -test.

Article Snippet: The cDNA of human ALKBH1 (GenBank NM_006020.2) was obtained by nested polymerase chain reaction from total RNA of HEK293T cells using specific primers ( ) and cloned into pE-SUMO-NSUN3 ( ) using the In-Fusion HD cloning kit (Clontech) to yield pE-SUMO-ALKBH1.

Techniques: In Vitro, Modification, Methylation, Mutagenesis, Incubation

Journal: Nucleic Acids Research

Article Title: ALKBH1 is an RNA dioxygenase responsible for cytoplasmic and mitochondrial tRNA modifications

doi: 10.1093/nar/gkx354

Figure Lengend Snippet: Genetic analyses of hm 5 Cm34/f 5 Cm34 formation in ct-tRNA Leu (CAA) ( A ) Schematic depiction of the human ALKBH1 gene and target site of mutations introduced by the CRISPR–Cas9 system. Shaded and open boxes indicate coding regions and untranslated regions of exons, respectively. Lines indicate introns. The target sequence of sgRNA in exon 4 is underlined, and the proto-spacer adjacent motif (PAM) sequence is boxed. Sequences of both alleles in KO1 and KO2 cell lines are aligned. Deleted nucleotides are indicated as dashed lines, and the inserted nucleotide is indicated by a gray letter. ( B ) Mass chromatograms of the anticodon-containing fragments of ct-tRNA Leu (CAA) containing f 5 Cm34 (top), hm 5 Cm34 (middle) and m 5 Cm34 (bottom) isolated from WT (left) and ALKBH1 KO (right) cell lines. n.d., not detected. Target peaks are indicated by gray triangles. Filled diamond in the left middle panel indicates a trace of an isotopic ion of f 5 Cm-containing fragment. Asterisk indicates an unidentified fragment whose molecular mass differs from that of the target fragment. ( C ) Schematic depiction of the human FTSJ1 gene and target site of the mutation introduced by the CRISPR–Cas9 system. Shaded and open boxes indicate coding regions and untranslated regions of exons, respectively. Lines indicate introns. The target sequence of sgRNA in exon 2 is underlined, and the PAM sequence is boxed. Sequences of both alleles in KO1 cell lines are aligned. Deleted nucleotides are indicated by dashed lines. ( D ) Mass chromatograms of the anticodon-containing fragments of ct-tRNA Leu (CAA) having f 5 Cm34 (top), hm 5 Cm34 (second), f 5 C34 (third) and hm 5 C34 (bottom) isolated from WT (left) and FTSJ1 KO (right) cell lines. n.d., not detected. Target peaks are indicated by gray triangles. Filled diamond in the left second panel indicates a trace isotopic ion of the f 5 Cm-containing fragment. Asterisk indicates an unidentified fragment whose molecular mass differs from that of the target fragment. ( E ) Biosynthetic pathways of hm 5 Cm34 and f 5 Cm34 in ct-tRNA Leu (CAA). Initially, NSUN2 methylates C34 to form m 5 C34 in the presence of AdoMet. ALKBH1 hydroxylates m 5 C34 to form hm 5 C34, and then oxidizes hm 5 C34 to yield f 5 C34. These reactions require O 2 and 2-OG as substrates. Precursor ct-tRNA Leu (CAA) bearing hm 5 C34 or f 5 C34 is exported to the cytoplasm and methylated by FTSJ1 together with a partner protein (probably WDR6) in an AdoMet-dependent manner to form hm 5 Cm34 or f 5 Cm34, respectively. In ALKBH1 KO cells (shaded), ct-tRNA Leu (CAA) containing m 5 C34 is exported to the cytoplasm, where it is methylated by FTSJ1 to form m 5 Cm34.

Article Snippet: The cDNA of human ALKBH1 (GenBank NM_006020.2) was obtained by nested polymerase chain reaction from total RNA of HEK293T cells using specific primers ( ) and cloned into pE-SUMO-NSUN3 ( ) using the In-Fusion HD cloning kit (Clontech) to yield pE-SUMO-ALKBH1.

Techniques: CRISPR, Sequencing, Isolation, Mutagenesis, Methylation

Journal: Nucleic Acids Research

Article Title: ALKBH1 is an RNA dioxygenase responsible for cytoplasmic and mitochondrial tRNA modifications

doi: 10.1093/nar/gkx354

Figure Lengend Snippet: ALKBH1 is essential for f 5 C34 biogenesis in mt-tRNA Met . ( A ) Mass chromatograms of the anticodon-containing fragments of mt-tRNA Met containing f 5 C34 (top), hm 5 C34 (second), m 5 C34 (third) and C34 (bottom) isolated from WT, ALKBH1 KO cell lines and KO1 rescued with plasmid-encoded ALKBH1 . n.d., not detected. Filled diamonds in the second panels indicate a trace isotopic ion of the f 5 C-containing fragment. ( B ) Biosynthetic pathway of f 5 C34 in mt-tRNA Met . NSUN3 methylates C34 to form m 5 C34 in the presence of AdoMet. ALKBH1 hydroxylates m 5 C34 to form hm 5 C34, and then oxidizes hm 5 C34 to yield f 5 C34. These reactions require O 2 and 2-OG as substrates.

Article Snippet: The cDNA of human ALKBH1 (GenBank NM_006020.2) was obtained by nested polymerase chain reaction from total RNA of HEK293T cells using specific primers ( ) and cloned into pE-SUMO-NSUN3 ( ) using the In-Fusion HD cloning kit (Clontech) to yield pE-SUMO-ALKBH1.

Techniques: Isolation, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: ALKBH1 is an RNA dioxygenase responsible for cytoplasmic and mitochondrial tRNA modifications

doi: 10.1093/nar/gkx354

Figure Lengend Snippet: ALKBH1 is required for mitochondrial function. ( A ) Growth curves of WT, ALKBH1 KO and NSUN3 KO cells in a medium containing glucose (left panel) or galactose (right panel) as a primary carbon source. Mean values of five independent cultures are plotted; error bars indicate s.d. ( B ) Relative activities of respiratory chain complexes in WT, ALKBH1 KO and NSUN3 KO cells. Activity of each complex is normalized against the corresponding citrate synthase (CS) activity. Data are presented as mean ± SD. of three independent assays. * P < 0.05, Student's t -test. ( C ) Oxygen consumption rate of WT, ALKBH1 KO and NSUN3 KO cells, measured by an XF24 extracellular flux analyzer. Data are presented as mean ± SD. of three independent assays. * P < 0.005, Student's t -test. ( D ) Pulse labeling of mitochondrial protein synthesis in WT, ALKBH1 KO and NSUN3 KO cells. Under inhibition of cytoplasmic translation, mitochondrial protein synthesis was labeled with [ 35 S] Met and [ 35 S] Cys.

Article Snippet: The cDNA of human ALKBH1 (GenBank NM_006020.2) was obtained by nested polymerase chain reaction from total RNA of HEK293T cells using specific primers ( ) and cloned into pE-SUMO-NSUN3 ( ) using the In-Fusion HD cloning kit (Clontech) to yield pE-SUMO-ALKBH1.

Techniques: Activity Assay, Labeling, Inhibition

Journal: Nucleic Acids Research

Article Title: ALKBH1 is an RNA dioxygenase responsible for cytoplasmic and mitochondrial tRNA modifications

doi: 10.1093/nar/gkx354

Figure Lengend Snippet: Level of m 1 A in mt-tRNAs increased in ALKBH1 KO cells ( A ) Mass chromatograms detecting negative ions ( z = −4 to −6) of RNase T 1 –digested fragments containing A16 (upper) and m 1 A16 (lower) of mt-tRNA Arg isolated from WT and ALKBH1 KO cells. ( B ) Mass chromatograms detecting negative ions ( z = −6 to −8) of RNase T 1 –digested fragments containing A58 (upper) and m 1 A58 (lower) of mt-tRNA Lys isolated from WT and ALKBH1 KO cells. ( C ) Detection of m 1 A16 in mt-tRNA Arg from WT and ALKBH1 KO cells by primer extension. The primer is shown as a black line, and the extended cDNAs for detection of m 1 A16 and A16 are depicted as red and blue lines, respectively. ( D ) Detection of m 1 A58 in mt-tRNA Lys from WT and ALKBH1 KO cells by primer extension. The primer is shown as a black line, and the extended cDNAs for detection of m 1 A58 and A58 are depicted as red and blue lines, respectively.

Article Snippet: The cDNA of human ALKBH1 (GenBank NM_006020.2) was obtained by nested polymerase chain reaction from total RNA of HEK293T cells using specific primers ( ) and cloned into pE-SUMO-NSUN3 ( ) using the In-Fusion HD cloning kit (Clontech) to yield pE-SUMO-ALKBH1.

Techniques: Isolation

Journal: Bone Research

Article Title: DNA N 6 -methyladenine demethylase ALKBH1 enhances osteogenic differentiation of human MSCs

doi: 10.1038/boneres.2016.33

Figure Lengend Snippet: Primers for quantitative RT-PCR and ChIP-qPCR

Article Snippet: For ALKBH1 overexpression, lentiviruses expressing the human ALKBH1 gene were purchased from Genecopoeia.

Techniques: Quantitative RT-PCR

Journal: Bone Research

Article Title: DNA N 6 -methyladenine demethylase ALKBH1 enhances osteogenic differentiation of human MSCs

doi: 10.1038/boneres.2016.33

Figure Lengend Snippet: ALKBH1 is upregulated during osteogenic differentiation. ( a ) Real-time RT-PCR of ALKBH1.n =3. * P <0.05 and ** P <0.01. ( b ) Western blot analysis.

Article Snippet: For ALKBH1 overexpression, lentiviruses expressing the human ALKBH1 gene were purchased from Genecopoeia.

Techniques: Quantitative RT-PCR, Western Blot

Journal: Bone Research

Article Title: DNA N 6 -methyladenine demethylase ALKBH1 enhances osteogenic differentiation of human MSCs

doi: 10.1038/boneres.2016.33

Figure Lengend Snippet: Depletion of ALKBH1 inhibits osteogenic differentiation. ( a ) Real-time RT-PCR . n =3. *** P <0.001. ( b ) Western blot analysis. ( c ) Representative images of dot blot assay. ( d ) Representative image of alkaline phosphatase (ALP) staining. ( e ) Quantitative analyses of the ALP activity. n =5. ** P <0.01. ( f ) Representative image of Alizarin red S (ARS) staining of MSCs. ( g ) Quantitative analyses of calcium mineralization. n =5. ** P <0.01. ( h – j ) Real-time RT-PCR revealed reduced RUNX2 , SP7 , and BGLAP messenger RNA expression. n =3. * P <0.05, ** P <0.01 and *** P <0.001.

Article Snippet: For ALKBH1 overexpression, lentiviruses expressing the human ALKBH1 gene were purchased from Genecopoeia.

Techniques: Quantitative RT-PCR, Western Blot, Dot Blot, Staining, Activity Assay, RNA Expression

Journal: Bone Research

Article Title: DNA N 6 -methyladenine demethylase ALKBH1 enhances osteogenic differentiation of human MSCs

doi: 10.1038/boneres.2016.33

Figure Lengend Snippet: Depletion of ALKBH1 inhibits bone formation in vivo . ( a ) Real-time RT-PCR . n =3. *** P <0.001. ( b ) Western blot analysis. ( c ) Representative images of dot blot assay. ( d ) Hematoxylin and eosin staining of the ectopic bone formation. ( e ) Quantitative analyses of bone volume versus total tissue volume (BV/TV). n =5. * P <0.05.

Article Snippet: For ALKBH1 overexpression, lentiviruses expressing the human ALKBH1 gene were purchased from Genecopoeia.

Techniques: In Vivo, Quantitative RT-PCR, Western Blot, Dot Blot, Staining

Journal: Bone Research

Article Title: DNA N 6 -methyladenine demethylase ALKBH1 enhances osteogenic differentiation of human MSCs

doi: 10.1038/boneres.2016.33

Figure Lengend Snippet: Overexpression of ALKBH1 enhances osteogenic differentiation. ( a ) Real-time RT-PCR . n =3. *** P <0.001. ( b ) Western blot analysis. ( c ) Representative images of dot blot assay. ( d ) Representative image of ALP staining. ( e ) Quantitative analyses of the ALP activity. n =5. * P <0.05. ( f ) Representative image of ARS staining of MSCs. ( g ) Quantitative analyses of calcium mineralization. n =5. *** P <0.001. ( h – j ) Real-time RT-PCR revealed increased messenger RNA expression of RUNX2 , SP7 , and BGLAP . n =3. * P <0.05 and *** P <0.001.

Article Snippet: For ALKBH1 overexpression, lentiviruses expressing the human ALKBH1 gene were purchased from Genecopoeia.

Techniques: Over Expression, Quantitative RT-PCR, Western Blot, Dot Blot, Staining, Activity Assay, RNA Expression

Journal: Bone Research

Article Title: DNA N 6 -methyladenine demethylase ALKBH1 enhances osteogenic differentiation of human MSCs

doi: 10.1038/boneres.2016.33

Figure Lengend Snippet: Depletion of ALKBH1 impairs ATF4 transcription. ( a ) Real-time RT-PCR showing decreased ATF4 expression . n =3. ** P <0.01. ( b ) Western blot analysis. ( c ) Chromatin immunoprecipitation (ChIP) assay for ALKBH1. ALKBH1 binds to the promoter region of ATF4 . n =4. ** P <0.01. ( d ) ChIP assay for N6-mA. Knockdown of ALKBH1 increases N6-mA levels on the promoter region of ATF4 . n =4. ** P <0.01.

Article Snippet: For ALKBH1 overexpression, lentiviruses expressing the human ALKBH1 gene were purchased from Genecopoeia.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Chromatin Immunoprecipitation