Journal: Nature Communications

Article Title: DOT1L safeguards cartilage homeostasis and protects against osteoarthritis

doi: 10.1038/ncomms15889

Figure Lengend Snippet: ( a ) Immunohistochemistry showing reduced methylated H3K79 levels (H3K79me2) that reflect loss of DOT1L activity in damaged areas from osteoarthritic patients (OA) as compared to their corresponding preserved areas and to cartilage from non-OA patients. Images are representative of images from four different patients. Scale bar, 400 μm. ( b ) Heat maps of differential mRNA expression determined by quantitative PCR in chondrocytes treated with DOT1L inhibitor EPZ-5676 (EPZ) or vehicle (V) from passage 0 (P0) until P2, and from preserved versus damaged areas in OA cartilage. The colour code represents the mean expression level of six and four independent patient samples respectively. ( c ) Immunoblot analysis showing decreased methylated H3K79 levels in mouse articular chondrocytes after intra-articular injection of EPZ into C57Bl/6 wild-type mouse knees. The image is representative of one experiment with protein extracts pooled from two or three mice per condition. Unprocessed original scans of blots are shown in . ( d , e ) C57/Bl6 wild-type mouse knees were injected with EPZ (5 mg kg –1 ) or vehicle and killed after 2 or 4 weeks. Knees were sectioned and stained with Hematoxylin-Safranin O ( d ). Scale bar, 200 μm. Cartilage damage was scored (see Methods section) and is shown in ( e ). One experiment was performed with n =10 and 5. Representative images from the 4 week evaluation are shown. * P <0.05 (two-tailed t -test). Error bars indicate mean±s.e.m.

Article Snippet: Antibody binding to the column was performed using 75 μg of either a mock antibody (donkey anti-goat IgG) as a control or DOT1L antibody (R&D Systems, MAB6546, clone 653613).

Techniques: Immunohistochemistry, Methylation, Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Injection, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: DOT1L safeguards cartilage homeostasis and protects against osteoarthritis

doi: 10.1038/ncomms15889

Figure Lengend Snippet: ( a ) KEGG pathway enrichment analysis of microarray data obtained from human articular chondrocytes treated with EPZ-5676 or vehicle. Nominal P values by EASE modified Fisher Exact test using the DAVID analysis tool (see Methods section) are shown. n =5 independent patient-derived cell cultures. ( b ) Co-immunoprecipitation (Co-IP) using an anti-DOT1L antibody showing interaction between DOT1L and β -catenin in human articular chondrocytes, that is increased upon Wnt activation by LiCl and disrupted upon DOT1L inhibition. The image is representative of three experiments. ( c ) TOP/FOP reporter assay in human articular chondrocytes after Wnt stimulation by LiCl and DOT1L inhibition by EPZ. Activity is compared to untreated cells (dotted line). n =3 biologically independent experiments. *** P <0.001 by one-way ANOVA. ( d , e ) LEF1 , TCF1 and c-MYC expression measured by quantitative PCR in chondrocytes treated with EPZ-5676 and LiCl ( d ) or in LiCl-treated chondrocytes transfected with siRNA directed against DOT1L or scrambled siRNA (siDOT1L or siSCR, respectively) ( e ). Data are from one experiment with three technical replicates. ( f ) Immunohistochemistry demonstrating increased TCF1 levels in the articular cartilage of C57/Bl6 wild-type mice after injection of EPZ-5676. The images are representative of three different animals. Scale bar, 200 μm.

Article Snippet: Antibody binding to the column was performed using 75 μg of either a mock antibody (donkey anti-goat IgG) as a control or DOT1L antibody (R&D Systems, MAB6546, clone 653613).

Techniques: Microarray, Modification, Derivative Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Activation Assay, Inhibition, Reporter Assay, Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Transfection, Immunohistochemistry, Injection

Journal: Nature Communications

Article Title: DOT1L safeguards cartilage homeostasis and protects against osteoarthritis

doi: 10.1038/ncomms15889

Figure Lengend Snippet: All experiments were performed in healthy human articular chondrocytes: treated as indicated with DOT1L inhibitor EPZ-5676, Wnt activator LiCl, SIRT1 antagonist EX527 or SIRT1 agonist SRT1720; or transfected with DOT1L or scrambled siRNA. All data are presented as mean±s.e.m. ( a ) Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) analysis of DOT1L and methylated H3K79 and ( b ) acetylated H3K9 (H3K9Ac) and methylated H3K4 (H3K4me3) as markers of active transcription on the transcriptional start site (TSS) of Wnt target genes. Data are from two to five experiments. ( c ) Expression levels of TCF1 Wnt target gene measured by quantitative PCR in chondrocytes transfected with indicated specific or scrambled siRNA (siSCR). Data are from one experiment with technical triplicates. ( d ) TCF1 expression measured by quantitative PCR in the presence of SIRT1 agonist and antagonist. Data from two experiments each with technical triplicates. ( e ) Co-IP analysis using the indicated antibodies demonstrating the interaction of DOT1L and SIRT1. The image is a representative image of three biologically independent experiments. ( f ) SIRT1 activity relative to vehicle-treated cells (dotted line). Data are from three biologically independent experiments. * P <0.05, *** P <0.001 by one-way ANOVA. ( g ) ChIP-qPCR analysis of SIRT1, PPARGC1A, GCN5 and EP300 binding on the TCF1 promoter and ( h ) TCF1 expression after siRNA transfection with indicated specific or scrambled siRNA. Data from two biologically independent experiments.

Article Snippet: Antibody binding to the column was performed using 75 μg of either a mock antibody (donkey anti-goat IgG) as a control or DOT1L antibody (R&D Systems, MAB6546, clone 653613).

Techniques: Transfection, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, ChIP-qPCR, Methylation, Expressing, Co-Immunoprecipitation Assay, Activity Assay, Binding Assay

Journal: Nature Communications

Article Title: DOT1L safeguards cartilage homeostasis and protects against osteoarthritis

doi: 10.1038/ncomms15889

Figure Lengend Snippet: ( a – c ) Inactivation of SIRT1 protects against DOT1L inhibitor-induced osteoarthritis: ( a ) C57/Bl6 wild-type mouse knees were injected with DOT1L inhibitor EPZ-5676 (5 mg kg −1 ) and SIRT1 inhibitor EX527 (1.25 mg kg –1 ), or vehicle (V) and killed after 4 weeks. Knees were sectioned and stained with Hematoxylin-Safranin O ( a ). Scale bar, 200 μm. Cartilage damage was scored (see Methods section) and is shown in ( b ). One experiment was performed with n =3 (vehicle), 8 (EPZ) and 10 (EPZ+EX527). * P <0.05 by one-way ANOVA. Error bars indicate mean±s.e.m. ( c ) Immunohistochemistry of TCF1 in the indicated groups. TCF1 levels are increased after EPZ treatment and normalized by additional EX527 treatment. The images are representative of three different animals. Scale bar, 200 μm. ( d , e ) Loss of DOT1L function causes severe growth retardation as demonstrated by skeletal staining ( d ) and histology of the growth plate ( e ) of 4-week-old Dot1l fl/fl ;Col2-Cre −/− (Cre-neg) and Dot1l fl/fl ;Col2-Cre +/− (Dot1l Cart-KO ) mice. ( f , g ) Increased TCF1 levels in Dot1l Cart-KO mice as shown by immunohistochemistry in the indicated mice strains in the articular cartilage ( f ) and growth plate ( g ). The images are representative of three different animals. Scale bar, 200 and 100 μm.

Article Snippet: Antibody binding to the column was performed using 75 μg of either a mock antibody (donkey anti-goat IgG) as a control or DOT1L antibody (R&D Systems, MAB6546, clone 653613).

Techniques: Injection, Staining, Immunohistochemistry

Journal: Nature Communications

Article Title: DOT1L safeguards cartilage homeostasis and protects against osteoarthritis

doi: 10.1038/ncomms15889

Figure Lengend Snippet: Upon Wnt signalling activation, DOT1L-containing complexes bind Wnt target gene chromatin. DOT1L interacts with SIRT1 and inhibits its function, preventing Wnt pathway hyper-activation. When Wnt signalling is activated in the absence of DOT1L function, high SIRT1 activity mediates the recruitment of transcriptional activators to LEF1 and TCF1 genes. High Wnt signalling leads to deleterious downstream effects and loss of cartilage homeostasis.

Article Snippet: Antibody binding to the column was performed using 75 μg of either a mock antibody (donkey anti-goat IgG) as a control or DOT1L antibody (R&D Systems, MAB6546, clone 653613).

Techniques: Activation Assay, Activity Assay

Journal: Cell Reports

Article Title: Aryl Hydrocarbon Receptor Contributes to the Transcriptional Program of IL-10-Producing Regulatory B Cells

doi: 10.1016/j.celrep.2019.10.018

Figure Lengend Snippet:

Article Snippet: Negative CD43- Isolation Kit , Miltenyi Biotec , Cat# 130-049-801.

Techniques: Western Blot, Recombinant, Methylation, Adjuvant, Enzyme-linked Immunosorbent Assay, Isolation, cDNA Synthesis, SYBR Green Assay, DNA Library Preparation, Purification, Bicinchoninic Acid Protein Assay, Microarray, Ex Vivo, Generated, Software, Red Blood Cell Lysis, Staining, Lysis

Journal: Advanced Science

Article Title: PRMT5 Enables Robust STAT3 Activation via Arginine Symmetric Dimethylation of SMAD7

doi: 10.1002/advs.202003047

Figure Lengend Snippet: PRMT5 potentiates STAT3 activation via Smad7. A) PRMT5 depletion dampens endogenous STAT3 activation in A549 cells. A549 cells stably expressing shPRMT5‐1 or shPRMT5‐2 or Control (shCtrl) were harvested and analyzed by using western blotting with indicated antibodies. B) Knockdown of PRMT5 attenuates IL‐6‐induced STAT3 phosphorylation in MCF10A cells. MCF10A cells were transfected with 40 pm siRNA against PRMT5. 36 h later, cells were treated with IL‐6 (10 ng mL −1 ) for the indicated time and harvested for western blotting analysis with appropriate antibodies. C) PRMT5 inhibition attenuates endogenous activation of STAT3 in H358 cells. H358 cells were treated with 20 × 10 −6 m of PRMT5 inhibitors EPZ015666 or GSK591 for the indicated time. Cell lysates were collected and subject to western blotting analysis. SDMA indicates global arginine di‐methylation. D) Smad7 potentiates STAT3 activation in A549 cells. A549 cells stably expressing FLAG‐GFP or FLAG‐Smad7 were harvested and subject to Western blotting analysis using appropriate antibodies. E) Stable knockdown of Smad7 dampens endogenous STAT3 activation in A549 cells. A549 cells stably expressing shSmad7 or shCtrl were harvested and subject to western blotting analysis using appropriate antibodies. F) Smad7 depletion dampens IL‐6‐induced STAT3 activation in MCF7 cells. Cells were transfected with siSmad7 (40 pm) and treated with IL‐6 (10 ng mL −1 ) for the indicated time. Cells were harvested and analyzed by western blotting with appropriate antibodies. G) Smad7 depletion dampens IL‐6‐induced STAT3 activation in MCF10A cells. Cell transfection, treatment, and Western blotting were done as described in Panel F. H) PRMT5 potentiates STAT3 activation dependent of Smad7. MCF10A cells were transduced with lentiviral particles expressing HA‐PRMT5 or HA‐G367A/R368A. After 24 h, cells were transfected with 40 pm siSmad7. 12 h later, cells were stimulated with IL‐6 (2 ng mL −1 ) for the indicated time. Cell lysates were harvested and subject to Western blotting analysis using appropriate antibodies.

Article Snippet: Antibodies and their commercial sources are as follows: PRMT5 (ab109451) and gp130 (ab202850) from Abcam; p‐STAT3 (9145), STAT3 (9139), HA (3724) and sdme‐RG (13222) from Cell Signaling Technology; SYM10 (07‐412) from Merck millipore; FLAG (F3165), β ‐actin (A5441), mouse IgG (I5381), and rabbit IgG(I5006) from Sigma‐Aldrich (USA); MYC (sc‐40) from Santa Cruz Biotechnology; Survivin (A19663) and c‐MYC (A1309) from Abclonal.

Techniques: Activation Assay, Stable Transfection, Expressing, Control, Western Blot, Knockdown, Phospho-proteomics, Transfection, Inhibition, Methylation, Transduction

Journal: Advanced Science

Article Title: PRMT5 Enables Robust STAT3 Activation via Arginine Symmetric Dimethylation of SMAD7

doi: 10.1002/advs.202003047

Figure Lengend Snippet: PRMT5 methylates Smad7 on R57. A) PRMT5 methylates Smad7. HEK293T cells were transfected with expression plasmids carrying MYC‐PRMT5/MEP50 and an SFB‐tagged construct, including gp130, JAK2, STAT3, Smad7, SHP2, and SOCS3. Cell lysates were harvested and precipitated with streptavidin beads. The retrieved complexes and input were analyzed by Western blotting with indicated antibodies. B) PRMT5 methylates the R57 residue on Smad7. HEK293T cells were transfected with MYC‐PRMT5/MEP50 and an SFB‐tagged Smad7 construct, i.e., wildtype Smad7 (WT) or a R‐to‐K substitution of Smad7 as indicated above the blots. Cell lysate was precipitated with streptavidin beads. Arginine di‐methylation of Smad7 was detected by Western blotting analysis. C) Mass spectrum of Smad7 Arg‐57 dimethylated peptide. Mass spectrometry identified Arg‐57 dimethylation of Smad7 in HEK293T cells expressing MYC‐PRMT5/MEP50 and SFB‐Smad7. Mass spectrometry profile of Smad7 sequence covering residue 47–64 is shown, and the dimethylated arginine side chains are indicated. D) PRMT5/MEP50 methylate Smad7, but not the R57K mutant. HEK293T cells were transfected MYC‐PRMT5/MEP50 and SFB‐Smad7 or Smad7 R57K mutant for 36 h. Cell lysates were harvested and immunoprecipitated with SYM10 antibody. The immunocomplexes and inputs were analyzed by Western blotting with indicated antibodies. E) PRMT5 methylates Smad7 in vitro. MYC‐PRMT5 or MYC‐G367A/R368A together with MYC‐MEP50 were immunopurified using anti‐MYC antibody from transfected HEK293T cells. Purified recombinant GST‐Smad7, GST‐Smad7 R57K mutant, and GST‐Smad4 were produced in E. coli . GST proteins and MYC‐PRMT5/MEP50 proteins were incubated in the presence of S‐adenosyl‐methionine to allow methylation reaction. Dimethylated Smad7 on R57 was detected by using Western blotting analysis.

Article Snippet: Antibodies and their commercial sources are as follows: PRMT5 (ab109451) and gp130 (ab202850) from Abcam; p‐STAT3 (9145), STAT3 (9139), HA (3724) and sdme‐RG (13222) from Cell Signaling Technology; SYM10 (07‐412) from Merck millipore; FLAG (F3165), β ‐actin (A5441), mouse IgG (I5381), and rabbit IgG(I5006) from Sigma‐Aldrich (USA); MYC (sc‐40) from Santa Cruz Biotechnology; Survivin (A19663) and c‐MYC (A1309) from Abclonal.

Techniques: Transfection, Expressing, Construct, Western Blot, Residue, Methylation, Mass Spectrometry, Sequencing, Mutagenesis, Immunoprecipitation, In Vitro, Purification, Recombinant, Produced, Incubation

Journal: Advanced Science

Article Title: PRMT5 Enables Robust STAT3 Activation via Arginine Symmetric Dimethylation of SMAD7

doi: 10.1002/advs.202003047

Figure Lengend Snippet: Arg methylation enhances Smad7 binding to gp130. A) Smad7 methylation increases its association with gp130. HEK293T cells were transfected with SFB‐Smad7 or Smad7 R57K mutant and HA‐gp130, together with MYC‐PRMT5/MEP50. Cell lysates were harvested and immunoprecipitated with Streptavidin beads. Western blotting analysis was done with appropriate antibodies. B) PRMT5 depletion blocks Smad7 methylation and its interaction with endogenous gp130. A549 tet‐on cells expressing SFB‐Smad7 were cultured with or without 1 µg mL −1 Dox for 3 d and then transfected with 40 × 10 −12 m siPRMT5. Cell lysates were harvested and immunoprecipitated with streptavidin beads. Endogenous gp130 was detected from the immunoprecipitates by using Western blotting analysis. C) Methylated Smad7 binds more tightly to gp130. HEK293T cells were transfected with indicated expression plasmids for MYC‐PRMT5, MYC‐G367A/R368A, and MYC‐MEP50 as well as SFB‐Smad7 or SFB‐R57K. Dimethylated Smad7 was immunopurified using SYM10 antibody, while total Smad7 was retrieved using an‐FLAG antibody. Bacterially expressed GST‐gp130‐ICD was purified using glutathione‐sepharose and eluted with elution buffer (10 × 10 −3 m glutathione, pH 8.0). In the in vitro binding experiments for evaluating the Smad7‐gp130 interaction, recombinant GST‐gp130‐ICD was added to the immunopurified Smad7. gp130‐ICD binding to immobilized Smad7 was analyzed by using Western blotting. D) Unmethylatable Smad7 R57K mutant loses its ability to potentiate STAT3 activation. MCF10A tet‐on cells stably expressing SFB‐Smad7 or Smad7 R57K were induced with 10 ng mL −1 Dox for 3 d, and treated with indicated concentrations of IL‐6. Cell lysates were collected and subject to Western blotting analysis.

Article Snippet: Antibodies and their commercial sources are as follows: PRMT5 (ab109451) and gp130 (ab202850) from Abcam; p‐STAT3 (9145), STAT3 (9139), HA (3724) and sdme‐RG (13222) from Cell Signaling Technology; SYM10 (07‐412) from Merck millipore; FLAG (F3165), β ‐actin (A5441), mouse IgG (I5381), and rabbit IgG(I5006) from Sigma‐Aldrich (USA); MYC (sc‐40) from Santa Cruz Biotechnology; Survivin (A19663) and c‐MYC (A1309) from Abclonal.

Techniques: Methylation, Binding Assay, Transfection, Mutagenesis, Immunoprecipitation, Western Blot, Expressing, Cell Culture, Purification, In Vitro, Recombinant, Activation Assay, Stable Transfection

Journal: Advanced Science

Article Title: PRMT5 Enables Robust STAT3 Activation via Arginine Symmetric Dimethylation of SMAD7

doi: 10.1002/advs.202003047

Figure Lengend Snippet: PRMT5 promotes STAT3 transcriptional and growth‐promoting responses. A) PRMT5 inhibition attenuates CDC25C expression in A549 cells. EPZ015666 or GSK591 (20 × 10 −6 m ) were added to A549 cells for 48 h. Cell lysates were harvested and analyzed by using qRT‐PCR to examine CDC25C mRNA levels. Data are shown as mean ± SD; n = 3. *** P < 0.001. B) PRMT5 inhibition attenuates CCNB1 expression in A549 cells. Cell treatment, harvest, and qRT‐PCR analysis were done as described in Panel A. Data are shown as mean ± SD; n = 3. *** P < 0.001. C) PRMT5 deficiency disables IL‐6/STAT3 responsiveness. GSEA showed that downregulated genes in PRMT5‐depleted A549 cells (shPRMT5‐2) were highly enriched in the IL‐6/STAT3 signaling gene set. Red, upregulated genes; blue, downregulated genes. NES = ‐1.73, FDR q value = 0.002. D) Heatmap showing expression levels (log 2 FPKM; left) and relative expression changes (log 2 (shPRMT5‐2/shCtrl); right) of the IL‐6/STAT3 signaling genes. E) Depletion of PRMT5 reduces DNA synthesis. A549 cells stably expressing shPRMT5‐1 or shPRMT5‐2 or Control (shCtrl) were subject to EdU staining to determine DNA incorporating rate (RiboBio),20x. F) Statistic analysis of the result in panel E. Data are shown as mean ± SD; n = 3. 0.01 < * P < 0.05. G) Inhibition of PRMT5 attenuates invasiveness in A549 cells. A549 cells were treated with PRMT5 inhibitors EPZ015666 or GSK591 (20 × 10 −6 m ) for 2 d, starved overnight in FBS‐free medium. 1 × 10 5 cells were plated in a transwell chamber and stained with crystal violet after 12 h. Purple color indicates crystal violet staining of the invaded cell population. H) PRMT5 depletion blocks colony formation. A549 stable cells were subject to crystal violet staining and photography.

Article Snippet: Antibodies and their commercial sources are as follows: PRMT5 (ab109451) and gp130 (ab202850) from Abcam; p‐STAT3 (9145), STAT3 (9139), HA (3724) and sdme‐RG (13222) from Cell Signaling Technology; SYM10 (07‐412) from Merck millipore; FLAG (F3165), β ‐actin (A5441), mouse IgG (I5381), and rabbit IgG(I5006) from Sigma‐Aldrich (USA); MYC (sc‐40) from Santa Cruz Biotechnology; Survivin (A19663) and c‐MYC (A1309) from Abclonal.

Techniques: Inhibition, Expressing, Quantitative RT-PCR, DNA Synthesis, Stable Transfection, Control, Staining

Journal: Advanced Science

Article Title: PRMT5 Enables Robust STAT3 Activation via Arginine Symmetric Dimethylation of SMAD7

doi: 10.1002/advs.202003047

Figure Lengend Snippet: PRMT5 promotes lung tumorigenesis. A) Depletion of PRMT5 attenuates tumorigenesis. LLC cells stably expressing shControl or mouse sh‐mPRMT5‐1 or sh‐mPRMT5‐2 were subcutaneously injected into female nude mice. Ten days after implantation, tumors were dissected and photographed. B) Measurement of tumor weight in Panel A. Data are shown as mean ± SD; n = 5 for each group. 0.01 < * P < 0.05. C) PRMT5 depletion impairs STAT3 signaling in tumors. Tumor samples were analyzed by Western blotting to examine phosphorylated STAT3 (p‐STAT3) and STAT3 target gene products such as c‐Myc and Survivin. D) PRMT5 is highly expressed in nonsmall cell lung cancer tissues (NSCLC). NSCLC tissue microarray (Alenabio) was subject to immunohistochemistry (Servicebio) using PRMT5 antibody. E) Statistic analysis of IHC score in Panel D. Statistical analysis was performed using a two‐tailed Student's t ‐test. Data are shown as mean ± SD. Lung cancer samples = 45. Normal lung tissue samples = 55. *** P < 0.001. F) A working model for PRMT5‐mediated STAT3 activation.

Article Snippet: Antibodies and their commercial sources are as follows: PRMT5 (ab109451) and gp130 (ab202850) from Abcam; p‐STAT3 (9145), STAT3 (9139), HA (3724) and sdme‐RG (13222) from Cell Signaling Technology; SYM10 (07‐412) from Merck millipore; FLAG (F3165), β ‐actin (A5441), mouse IgG (I5381), and rabbit IgG(I5006) from Sigma‐Aldrich (USA); MYC (sc‐40) from Santa Cruz Biotechnology; Survivin (A19663) and c‐MYC (A1309) from Abclonal.

Techniques: Stable Transfection, Expressing, Injection, Western Blot, Microarray, Immunohistochemistry, Two Tailed Test, Activation Assay

Journal: Current Biology

Article Title: Long-Fiber Carbon Nanotubes Replicate Asbestos-Induced Mesothelioma with Disruption of the Tumor Suppressor Gene Cdkn2a ( Ink4a/Arf )

doi: 10.1016/j.cub.2017.09.007

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (clone 197G2) , Cell Signaling Technology , Cat# 4377; RRID: AB_331775.

Techniques: Recombinant, Produced, Reverse Transcription, Microarray, Labeling, DNA Purification, Methylation, TA Cloning, Sequencing, SYBR Green Assay, Software

Journal: Cancers

Article Title: Aspartate-β-Hydroxylase: A Promising Target to Limit the Local Invasiveness of Colorectal Cancer

doi: 10.3390/cancers12040971

Figure Lengend Snippet: Bioinformatics analysis of aspartate-β-hydroxylase (ASPH) overexpression in CRC: ( a ) analysis of ASPH mRNA expression in normal mucosa and colorectal cancer (CRC) samples from TCGA database (UALCAN website); ( b ) ASPH promoter methylation status in normal mucosa and CRC samples from TCGA database (UALCAN website; beta-value scale ranges from 0 = unmethylated to 1 = fully methylated); ( c ) analysis of ASPH protein levels by mass spectrometry in normal mucosa and CRC samples from the CPTAC database (UALCAN website; Z-values represent standard deviations from the median across the samples); ( d ) putative copy number alterations of the ASPH gene computed by GISTIC algorithm in TCGA database (c-BioPortal website; shallow deletion = 1− copy, gain = 1+ copy, amplification ≥ 2+). Box plots represent the quartile distribution of data around the median value. Statistics are reported in each graph as the calculated p value.

Article Snippet: The anti-ASPH mouse monoclonal antibody (clone A10, IgG1, Santa Cruz, Santa Cruz, CA, USA) was raised against aminoacidic residues 382–681 (which are not shared with the truncated proteins Junctine, Junctate and Humbug, derived from the same gene [ ]) and specifically recognized ASPH in its active, high molecular weight form (86 KDa) ( ).

Techniques: Over Expression, Expressing, Methylation, Mass Spectrometry, Amplification

Journal: Cancers

Article Title: Aspartate-β-Hydroxylase: A Promising Target to Limit the Local Invasiveness of Colorectal Cancer

doi: 10.3390/cancers12040971

Figure Lengend Snippet: Heatmaps of mRNA-clusters significantly correlated with ASPH expression (red = high, green = low), selected in : ( a ) Immune signature ( n = 222); ( b ) Notch signature ( n = 203); ( c ) Invasive signature ( n = 203). Each cluster is ordered according to Pearson’s r coefficients of each marker against ASPH, from higher (left) to lower (right). The maximum r coefficient was 0.330 (SNW1), and the lowest was -0.304 (LFNG); thus, no marker showed a widespread correlation with ASPH among analyzed samples. Microarray Z -scores for the selected markers were extracted from the COAD Firehouse legacy database (TCGA) using the cBioPortal web interface.

Article Snippet: The anti-ASPH mouse monoclonal antibody (clone A10, IgG1, Santa Cruz, Santa Cruz, CA, USA) was raised against aminoacidic residues 382–681 (which are not shared with the truncated proteins Junctine, Junctate and Humbug, derived from the same gene [ ]) and specifically recognized ASPH in its active, high molecular weight form (86 KDa) ( ).

Techniques: Expressing, Marker, Microarray

Journal: Cancers

Article Title: Aspartate-β-Hydroxylase: A Promising Target to Limit the Local Invasiveness of Colorectal Cancer

doi: 10.3390/cancers12040971

Figure Lengend Snippet: ASPH expression in the tumor invasive margin (IM) correlates with the pattern of local invasion and patients’ overall survival: invasive front type (I = infiltrative, E = expansive), tumor budding (1 = 0–4 buds, 2 = 5–9 buds, 3 ≥ 10 buds) and overall survival (L = low, H = high ASPH levels dichotomized by the median value) show a strong relation only with ASPH levels scored in CRC IM. Center of the tumor (CT)-scored ASPH shows a statistically significant relation only with tumor budding, while ASPH expressed next to tumor lumen (LM) does not show any relation with these parameters. Box plots represent the quartile distribution of ASPH H-score quantifications in CRC samples around the median value. Statistics are reported in each graph as the calculated p value (Invasive front: Student t -test; Budding: one-way ANOVA; Overall survival: Kaplan–Meier analysis).

Article Snippet: The anti-ASPH mouse monoclonal antibody (clone A10, IgG1, Santa Cruz, Santa Cruz, CA, USA) was raised against aminoacidic residues 382–681 (which are not shared with the truncated proteins Junctine, Junctate and Humbug, derived from the same gene [ ]) and specifically recognized ASPH in its active, high molecular weight form (86 KDa) ( ).

Techniques: Expressing

Journal: Cancers

Article Title: Aspartate-β-Hydroxylase: A Promising Target to Limit the Local Invasiveness of Colorectal Cancer

doi: 10.3390/cancers12040971

Figure Lengend Snippet: ASPH silencing or activity inhibition decreases cell invasion and proliferation in a panel of CRC cell lines. Specific ASPH siRNAs visibly downregulate ASPH expression after 18 h ( a ). Concomitantly, cell invasion ( b ) and proliferation ( c ) are significantly inhibited. All these effects are associated with Notch1 inhibition ( d ). One representative experiment out of three is reported. β-Actin was utilized as a loading control. Means ± SEM are reported. *** p < 0.001. DLD1 and SW480 cell lines were exposed to increasing concentration of MO-I-1144. For the invasion test ( e ), filters were recovered and counted after 18 h. Representative images of filters from 20 µM treated cells are shown in ( f ). Cell growth was evaluated after 96 h by the crystal violet assay ( g ). One representative experiment (conducted on 10 replicates) out of three is reported. Mean ± SEM are reported. ** p < 0.01, *** p < 0.001.

Article Snippet: The anti-ASPH mouse monoclonal antibody (clone A10, IgG1, Santa Cruz, Santa Cruz, CA, USA) was raised against aminoacidic residues 382–681 (which are not shared with the truncated proteins Junctine, Junctate and Humbug, derived from the same gene [ ]) and specifically recognized ASPH in its active, high molecular weight form (86 KDa) ( ).

Techniques: Activity Assay, Inhibition, Expressing, Control, Concentration Assay, Crystal Violet Assay

Journal: Cell Reports Methods

Article Title: Engineering CpG island DNA methylation in pluripotent cells through synthetic CpG-free ssDNA insertion

doi: 10.1016/j.crmeth.2023.100465

Figure Lengend Snippet:

Article Snippet: Mouse α-MYH7 monoclonal , Santa Cruz Biotech , Cat# Sc-53090; RRID: AB_2147279.

Techniques: Recombinant, Membrane, Transfection, SYBR Green Assay, DNA Methylation Assay, Mutagenesis, CRISPR, Software, Functional Assay, Microarray, In Vivo, Comparison, Next-Generation Sequencing

Journal: International Journal of Biological Sciences

Article Title: Mitochondrial Dysfunction by FADDosome Promotes Gastric Mucosal Injury in Portal Hypertensive Gastropathy

doi: 10.7150/ijbs.90835

Figure Lengend Snippet: NOX2 enhances FADDosome-induced gastric epithelial mitochondrial fission and dysfunction in PHG. (A) Two-dimensional hierarchical clustering results for the enzyme NADPH oxidase (NOX) family were presented for the comparison between PHG patients and healthy volunteers (n = 3 per group). The fold changes and P values of the indicated mRNAs in PHG tissues relative to those in normal (Uninvolved) tissues from the microarray experiment were also presented. (B) NOX1, NOX2 and NOX4 IHC staining (brown) showed that NOX2, but not NOX1 or NOX4, was upregulated in the gastric mucosal samples of PHG patients, and the NOX1, NOX2 and NOX4 areas were also analyzed. n = 6 per group. * P < 0.05. (C) Representative images of IHC staining (brown) for NOX1, NOX2 and NOX4 in mouse models revealed that Nec-1 (an inhibitor of RIPK1) repressed NOX2 expression in mice with PVL. n = 6 per group. (D) The NOX1, NOX2 and NOX4 areas detected by IHC staining in (C) were presented. n = 6 per group. * P < 0.05 versus SO mice; # P < 0.05 versus mice with PVL without Nec-1 treatment. (E) The colocalization of TOMM20 (red) and NOX2 (green) determined by confocal imaging in GES-1 cells confirmed that TNF-α increased the mitochondrial localization of NOX2 (white arrows indicate colocalization). Nuclei (blue) were counterstained with DAPI. (F) The levels of and interaction between NOX2 and TOMM20 were determined by western blotting, and IP showed that Nec-1 decreased the epithelial expression of total and mitochondrial NOX2 in the primary epithelial cells of mice with PVL. TEM also revealed that Nec-1 partly normalized the abnormal or damaged mitochondria in the mice with PVL. n = 6 per group. * P < 0.05 versus SO mice; # P < 0.05 versus mice with PVL without Nec-1 treatment. (G) The ATP concentration in the mouse models verified that Nec-1 and GSK2795039 (GSK, a NOX2 inhibitor) improved the production of ATP in mice with PVL. n = 6 per group. * P < 0.05. (H) Representative 4-HNE IHC staining (brown) and quantification analysis in SO mice and mice with PVL suggested that GSK2795039 (GSK) reduced the level of 4-HNE in mice with PVL. n = 6 per group. * P < 0.05 versus SO mice; # P < 0.05 versus mice with PVL without GSK2795039 treatment. (I) Representative MitoSox staining (red) and the MitoSox intensity in mouse model revealed that GSK2795039 alleviated mitochondrial ROS (mtROS) accumulation in the primary epithelial cells of mice with PVL. n = 6 in each group. * P < 0.05 versus SO mice; # P < 0.05 versus mice with PVL without GSK2795039 treatment. (J) Related protein levels in primary epithelial cells from mouse models treated with the indicated agents ( shFADD , Nec-1 or BAY) showed interactions among TAK1, NF-κBp65, FADD and NOX2 signaling. (K) GES-1 cells were transfected with either the control vector or shFADD and then treated with TNF-α or vehicle, and the NOX2 luciferase reporter activities were presented (left panel). * P < 0.05 versus the vehicle group and TNF-α-treated cells transfected with shFADD . The NOX2 luciferase reporter activities in GES-1 cells transfected with pcDNA3.1-p65 -vector or pcDNA3.1 -vector were also shown (right panel), * P < 0.05.

Article Snippet: In some experiments, the mice were treated for 2 weeks after the operation with the following reagents or drugs according to the experimental needs: for NF-κB inhibition, the mice were intraperitoneally injected with Bay11708 (BAY, Calbiochem, La Jolla, CA, USA) at 200 μg/per mouse daily for 2 weeks; for TNF-α inhibition, the mice were intraperitoneally injected with infliximab (IFX, Janssen Biotech, Horsham, PA, USA) at 5 mg/kg per day for 2 weeks; for RIPK1 inhibition, the mice were intraperitoneally injected with 2 mg/kg necrostatin-1 (Nec-1, HY-15760, MedChemExpress, NJ, USA) per day for 2 weeks; for above experiments of the inhibitors, the mice in the control group (vehicle group) were intraperitoneally injected with an equal volume of phosphate-buffered saline (PBS) daily for 2 weeks; for Drp1 inhibition, the mice were intraperitoneally injected with 20 mg/kg Mdivi-1 (dissolved in DMSO, HY-15886, MedChemExpress), and the control animals (vehicle) were injected with an equal volume of DMSO; for ROS scavenging, the mice were intraperitoneally injected with Mito-TEMPO (MT, dissolved in PBS, 10 mg/kg, HY-112879, MedChemExpress) every other day for 2 weeks, and the mice in the control group (vehicle) were intraperitoneally injected with an equal volume of PBS; for NOX2 blockade, 50 mg/kg GSK2795039 (GSK, dissolved in 20% DMSO, 20% Tween 80 and 60% polyethylene glycol 200, HY-18950, MedChemExpress) was administered by intraperitoneal injection daily for 2 weeks; for pan-caspase inhibition, the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD-FMK, dissolved in 10% DMSO, 40% PEG300, 5% Tween-80 and 45% saline, 10 mg/kg daily; HY-16658B; MedChemExpress) was administered by intraperitoneal injection for 2 weeks, and the mice in the control group (vehicle) were intraperitoneally injected with an equal volume of the abovementioned solvents daily for 2 weeks.

Techniques: Comparison, Microarray, Immunohistochemistry, Expressing, Imaging, Western Blot, Concentration Assay, Staining, Transfection, Control, Plasmid Preparation, Luciferase

Journal: International Journal of Biological Sciences

Article Title: Mitochondrial Dysfunction by FADDosome Promotes Gastric Mucosal Injury in Portal Hypertensive Gastropathy

doi: 10.7150/ijbs.90835

Figure Lengend Snippet: The alteration of glycolysis associated with dysfunction of the mitochondrial electron transport chain contributes to oxidative stress in the PHG. (A) Western blotting showed that the protein levels of LDHA and HK2 were increased in mice with PVL but were decreased by GSK2795039 (GSK, a NOX2 inhibitor) in mice with PVL. The ratios of the normalized LDHA/β-actin and HK2/β-actin densitometric units and the relative level of lactic acid were also analyzed. n = 6 per group. *P < 0.05 versus SO mice; #P < 0.05 versus mice with PVL not treated with GSK2795039. (B) Oxygen consumption rate (OCR) analysis of primary epithelial cells isolated from mouse models suggested that increased nonmitochondrial oxygen consumption and reduced maximum respiration were present in mice with PVL but were reversed by GSK2795039. n = 6 in each group. * P < 0.05 versus SO mice; # P < 0.05 versus mice with PVL without GSK2795039 treatment. (C) Examination of the glycolytic rate (for the extracellular acidification rate [ECAR]) showed that GSK2795039 treatment reduced basal and compensatory glycolysis in mice with PVL. n = 6 in each group. * P < 0.05 versus SO mice; # P < 0.05 versus mice with PVL without GSK2795039 treatment. (D) Two-dimensional hierarchical clustering results of the genes of electron transport chain (ETC)-related elements between PHG patients (as PHG) and healthy volunteers (as Uninvolved) (n = 3 per group). (E) Western blotting analysis (left panel) of protein levels of representative ETC complex subunits in the primary epithelial cells of PHG patients and healthy volunteers (as Uninvolved) revealed that the levels of the mitochondrial complex I, II, III, IV and V subunits were decreased in PHG patients. The activity of the antioxidant enzyme SOD and the ratio of reduced (GSH) to oxidized (GSSG) states (GSH/GSSG) detected by assay kits (right panel) were found to be decreased in PHG. n = 6 per group. * P < 0.05. (F) Western blotting analysis of representative ETC complex subunits from primary epithelial cells isolated from mouse models suggested that Nec-1 (an inhibitor of RIPK1) mitigated the defects in ETC subunit expression and decreased ROS levels in mice with PVL. The ratios of the normalized NDUFA9/β-actin, SDHA/β-actin, Cyt b/β-actin, COX I/β-actin and ATP5A/β-actin densitometric units and the levels of ROS were also analyzed. n = 6 per group. *P < 0.05 versus SO mice; #P < 0.05 versus mice with PVL without Nec-1 treatment. (G) SOD activity and the GSH/GSSG ratio, as detected by assay kits, were inhibited in mice with PVL but were restored by Nec-1 treatment. n = 6 per group. *P < 0.05 versus SO mice; #P < 0.05 versus mice with PVL without Nec-1 treatment. (H) Western blotting analysis and quantification of protein levels for representative ETC complex subunits from primary epithelial cells isolated from mouse models showed that Mdivi-1 reversed the decrease in mitochondrial complex I, II, III, IV and V subunit levels and reduced ROS levels in mice with PVL. The ratios of the normalized NDUFA9/β-actin, SDHA/β-actin, Cyt b/β-actin, COX I/β-actin and ATP5A/β-actin densitometric units and the levels of ROS were also presented. n = 6 per group. *P < 0.05 versus SO mice; #P < 0.05 versus mice with PVL without Mdivi-1 treatment. (I) Decreases in SOD activity and the GSH/GSSG ratio detected by assay kits were found in mice with PVL but were reversed by Mdivi-1 treatment. n = 6 per group. *P < 0.05 versus SO mice; #P < 0.05 versus mice with PVL without Mdivi-1 treatment.

Article Snippet: In some experiments, the mice were treated for 2 weeks after the operation with the following reagents or drugs according to the experimental needs: for NF-κB inhibition, the mice were intraperitoneally injected with Bay11708 (BAY, Calbiochem, La Jolla, CA, USA) at 200 μg/per mouse daily for 2 weeks; for TNF-α inhibition, the mice were intraperitoneally injected with infliximab (IFX, Janssen Biotech, Horsham, PA, USA) at 5 mg/kg per day for 2 weeks; for RIPK1 inhibition, the mice were intraperitoneally injected with 2 mg/kg necrostatin-1 (Nec-1, HY-15760, MedChemExpress, NJ, USA) per day for 2 weeks; for above experiments of the inhibitors, the mice in the control group (vehicle group) were intraperitoneally injected with an equal volume of phosphate-buffered saline (PBS) daily for 2 weeks; for Drp1 inhibition, the mice were intraperitoneally injected with 20 mg/kg Mdivi-1 (dissolved in DMSO, HY-15886, MedChemExpress), and the control animals (vehicle) were injected with an equal volume of DMSO; for ROS scavenging, the mice were intraperitoneally injected with Mito-TEMPO (MT, dissolved in PBS, 10 mg/kg, HY-112879, MedChemExpress) every other day for 2 weeks, and the mice in the control group (vehicle) were intraperitoneally injected with an equal volume of PBS; for NOX2 blockade, 50 mg/kg GSK2795039 (GSK, dissolved in 20% DMSO, 20% Tween 80 and 60% polyethylene glycol 200, HY-18950, MedChemExpress) was administered by intraperitoneal injection daily for 2 weeks; for pan-caspase inhibition, the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD-FMK, dissolved in 10% DMSO, 40% PEG300, 5% Tween-80 and 45% saline, 10 mg/kg daily; HY-16658B; MedChemExpress) was administered by intraperitoneal injection for 2 weeks, and the mice in the control group (vehicle) were intraperitoneally injected with an equal volume of the abovementioned solvents daily for 2 weeks.

Techniques: Western Blot, Isolation, Activity Assay, Expressing

Journal: Nucleic acids research

Article Title: A distinct class of eukaryotic MT-A70 methyltransferases maintain symmetric DNA N6-adenine methylation at the ApT dinucleotides as an epigenetic mark associated with transcription.

doi: 10.1093/nar/gkz1053

Figure Lengend Snippet: Figure 1. Phylogenetic analysis and domain structure comparison of AMTs 1–7. (A) Phylogenetic analysis of MT-A70 proteins. DNA 6mA (subclades AMT2/5, AMT1, METTL4/DAMT1 and AMT6/7) and RNA m6A (subclades METTL3 and METTL14) methyltransferase candidates are separated by a dotted line. Species are marked by different colors based on their phylogenetic position in the eukaryotic tree (inset). AMTs 1–7 of Tetrahymena are in bold plus red. The scale bar corresponds to 1 expected amino acid substitution per site. See Supplementary Table S9 for details (species full name and NCBI GI number). (B) Conserved domains and motifs in AMTs 1–7. Gene names used in Luo et al. (70) and Beh et al. (75) are shown in parentheses. MT-A70 domains of AMTs 1–5 were predicted by CD-Search (69), while domain structures of AMT6 and AMT7 were inferred from sequence alignment with AMTs 1–5.

Article Snippet: After crosslink by 3% PFA, -6mA (Synaptic Systems, 202003, 1:2000) and its secondary antibody (goat anti-rabbit IgG (H+L), Alexa Fluor 488, Invitrogen, A-11008, 1:4000) were used.

Techniques: Comparison, Sequencing

Journal: Nucleic acids research

Article Title: A distinct class of eukaryotic MT-A70 methyltransferases maintain symmetric DNA N6-adenine methylation at the ApT dinucleotides as an epigenetic mark associated with transcription.

doi: 10.1093/nar/gkz1053

Figure Lengend Snippet: Figure 2. Germline knockout of AMT1 (AMT1) dramatically reduced the DNA 6mA level. (A) Immunofluorescence (IF) staining of DNA 6mA in logarithmically growing WT and AMT1 cells. Note the absence of 6mA signals in the MIC (arrowheads). (B) Statistical analysis of 6mA IF signal intensity in A. Cell images were randomly selected (WT: n = 263, AMT1: n = 450) and processed by ImageJ. Data are presented as box plots (from top to bottom: max, first quartile, median, third quartile, and min). Student’s t-test was performed. ***P < 0.001. (C) Mass spectrometry analysis of 6mA, performed on five biological replicates for WT and 39 for AMT1. Data are presented as box plots. Student’s t-test was performed. ***P < 0.001. (D) Expression profile of AMT1 during growth (mid-log phase, ∼3.5 × 105 cells/ml), starvation (15 h after starvation) and conjugation (4 h post-mixing). Expression levels are represented by normalized RNA-seq reads numbers (http://tfgd.ihb.ac.cn/) (88), microarray signals (31) and quantitative RT-PCR data. All expression values (in logarithm scale) were compared with values during starvation (set at 0). (E) IF staining of DNA 6mA in conjugating WT and AMT1 cells. Nuclear events are used to ascertain conjugation stages (see schematics on the left). The new MAC is outlined (dotted circles). Note that de novo occurrence of 6mA in the new MAC was dramatically reduced in AMT1 cells. (F) IF staining of AMT1 in growing cells. AMT1 was HA-tagged at the N-terminus (HA-AMT1) and over-expressed under the control of the MTT1 promoter. After induction by cadmium chloride (1.5g/ml, 30min), HA-AMT1 was detected by an -HA antibody. Note the absence of AMT1 signals in the MIC (arrowheads). (G) IF staining of AMT1 in conjugating cells. Nuclear events are used to ascertain conjugation stages (see schematics on the left). Note that AMT1 signals appeared in the new MAC (dotted circles), before buildup of 6mA signals.

Article Snippet: After crosslink by 3% PFA, -6mA (Synaptic Systems, 202003, 1:2000) and its secondary antibody (goat anti-rabbit IgG (H+L), Alexa Fluor 488, Invitrogen, A-11008, 1:4000) were used.

Techniques: Knock-Out, Immunofluorescence, Staining, Mass Spectrometry, Expressing, Conjugation Assay, RNA Sequencing, Microarray, Quantitative RT-PCR, Control

Journal: Nucleic acids research

Article Title: A distinct class of eukaryotic MT-A70 methyltransferases maintain symmetric DNA N6-adenine methylation at the ApT dinucleotides as an epigenetic mark associated with transcription.

doi: 10.1093/nar/gkz1053

Figure Lengend Snippet: Figure 3. Growth and development were severely impaired in AMT1 loss-of-function mutants. (A) Growth rates of WT (SB210), AMT1, AMT1-APPA and AMT1-rescued cells (AMT1-RS). Cells were enumerated using a Coulter counter at the indicated time points. Doubling time (h) were calculated based on the log-phase data. (B) Trajectory analysis of the swimming ability. Swimming paths of indicated cells in half a second were recorded and converted to illustrations by Image J. AMT1 and AMT1-APPA cells swam much slower than WT cells. The swimming ability was restored in AMT1-RS cells. (C) Quantification of the translational swimming velocity. All values are normalized with that of WT cells. Data are presented as mean ± standard deviations. Student’s t-test was performed. ***P < 0.001; ns: not significant (P > 0.05); compared with WT, unless indicated otherwise. (D) Contractile vacuoles (CVs) observed by phase-contrast microscopy. Abnormal CVs (red arrowheads) were observed in a large portion of AMT1 and AMT1-APPA cells, but not in WT and AMT1-RS cells. (E) IF staining of 6mA in WT, AMT1, AMT1-APPA and AMT1-RS cells. Note the absence of 6mA signal in the MIC (white arrowheads). (F) Mass spectrometry analysis of 6mA in WT, AMT1, AMT1-APPA and AMT1-RS cells (two independent strains). Data are presented as mean ± standard deviations. Student’s t-test was performed. ***P < 0.001; ns: not significant (P > 0.05); compared with WT, unless indicated otherwise. (G) Correlations in gene expression profiles. Pearson’s correlation coefficients (PCC) were calculated for pair-wise comparison and represented by the color scale. Cluster analysis, based on PCC, is shown on the left. Note the similarities between the gene expression profiles of WT and AMT1-rescued cells (AMT1-RS-14 and AMT1-RS-17) and their distinction from that of AMT1 cells. (H) Conjugation progress (h) of WT (SB210 × CU428) and AMT1 (1–10 × 1–11) cells. Cells at each time point (n > 200) were classified into different developmental stages by their nuclear morphology: pre-meiosis (E1), meiosis (E2), mitosis (M), new MAC development (L1) and pair separation (L2) (89).

Article Snippet: After crosslink by 3% PFA, -6mA (Synaptic Systems, 202003, 1:2000) and its secondary antibody (goat anti-rabbit IgG (H+L), Alexa Fluor 488, Invitrogen, A-11008, 1:4000) were used.

Techniques: Microscopy, Staining, Mass Spectrometry, Gene Expression, Comparison, Conjugation Assay

Journal: Nucleic acids research

Article Title: A distinct class of eukaryotic MT-A70 methyltransferases maintain symmetric DNA N6-adenine methylation at the ApT dinucleotides as an epigenetic mark associated with transcription.

doi: 10.1093/nar/gkz1053

Figure Lengend Snippet: Figure 4. Deletion of AMT1 affected the genome-wide distribution of 6mA. (A) 6mA density (6mA/A) in WT (blue) and AMT1 cells (red), across the assembled Tetrahymena MAC genome, which are concatenated according to their positions in the MIC chromosomes. (B) Classification of 6mA sites according to their methylation levels (left half) and symmetry (right half), in WT (blue) and AMT1 cells (red). The left and right panels represent per- centage (a particular class of 6mA/all 6mA) and the site numbers for the classes, respectively. See Supplementary Table S3 for details. (C) Normalized 6mA distribution on scaffold 8254803 (the longest in the Tetrahymena MAC genome assembly) in WT and AMT1 cells. Classification of 6mA sites are the same as in (B). (D) Sequence logos for 6mA sites (at position 0) in WT and AMT1 cells. (E) Relative distribution of 6mA in ApN (ApA/ApT/ApG/ApC)

Article Snippet: After crosslink by 3% PFA, -6mA (Synaptic Systems, 202003, 1:2000) and its secondary antibody (goat anti-rabbit IgG (H+L), Alexa Fluor 488, Invitrogen, A-11008, 1:4000) were used.

Techniques: Genome Wide, Methylation, Sequencing

Journal: Nucleic acids research

Article Title: A distinct class of eukaryotic MT-A70 methyltransferases maintain symmetric DNA N6-adenine methylation at the ApT dinucleotides as an epigenetic mark associated with transcription.

doi: 10.1093/nar/gkz1053

Figure Lengend Snippet: Figure 5. AMT1 affects Pol II-transcribed genes. (A) Distributions of 6mA, H3K4me3, H2A.Z, and nucleosomes. In this GBrowse snapshot of a represen- tative genomic region, tracks from top to bottom are: gene models, mRNA transcripts, 6mA (WT in blue, AMT1 in red), H3K4me3 (X-ChIP coverage), dyads of nucleosomes containing H2A.Z, and dyads of nucleosomes (WT in blue, AMT1 in red). Note the biased distribution of 6mA toward the 5′ end of a long gene (TTHERM 00498010). (B) Validating the methylation states of 10 GATC sites by DpnI/DpnII digestion. The sites were selected for their location on genes transcribed by different RNA polymerases (Pol I, II and III), and their methylation levels calculated from SMRT sequencing data

Article Snippet: After crosslink by 3% PFA, -6mA (Synaptic Systems, 202003, 1:2000) and its secondary antibody (goat anti-rabbit IgG (H+L), Alexa Fluor 488, Invitrogen, A-11008, 1:4000) were used.

Techniques: Methylation, Sequencing

Journal: Nucleic acids research

Article Title: A distinct class of eukaryotic MT-A70 methyltransferases maintain symmetric DNA N6-adenine methylation at the ApT dinucleotides as an epigenetic mark associated with transcription.

doi: 10.1093/nar/gkz1053

Figure Lengend Snippet: Figure 6. Attenuation of strong 6mA and nucleosome positioning relative to each other in AMT1 cells. (A) Distribution profiles of 6mA (top panel) and nucleosome (bottom panel) around TSS in WT (blue) and AMT1 cells (red). (B) Nucleosome positioning in WT (blue) and AMT1 (red) cells. Degrees of positioning were calculated for +1, +2 and +3 nucleosomes in the gene body. See Materials and Methods for details. (C) 6mA distribution relative to the nucleosome dyad in WT (top panel) and AMT1 cells (bottom panel). The violin plots show the density of 6mA between neighboring nucleosome dyads, grouped by methylation levels. The box plots within represent the median and the interquartile range of each group. Red dotted lines mark the trend for 6mA with high methylation levels to be enriched in linker DNA in WT (top panel) but not AMT1 cells (bottom panel). (D) Dispersions of 6mA increase further downstream of TSS in AMT1 cells (right panel) but not WT cells (left panel). The violin/box plots show 6mA distribution between neighboring nucleosomes, grouped by their positions in the gene body (+1/+2, +2/+3, +3/+4, +4/+5).

Article Snippet: After crosslink by 3% PFA, -6mA (Synaptic Systems, 202003, 1:2000) and its secondary antibody (goat anti-rabbit IgG (H+L), Alexa Fluor 488, Invitrogen, A-11008, 1:4000) were used.

Techniques: Methylation

Journal: Nucleic acids research

Article Title: A distinct class of eukaryotic MT-A70 methyltransferases maintain symmetric DNA N6-adenine methylation at the ApT dinucleotides as an epigenetic mark associated with transcription.

doi: 10.1093/nar/gkz1053

Figure Lengend Snippet: Figure 7. AMT1 represents a new class of DNA 6mA methyltransferases and epigenetic regulators in eukaryotes. (A) A diagram summarizing AMT1′s roles as an epigenetic regulator. (B) Phyletic distribution of MT-A70 family genes (AMT1, AMT6/7, and METTL4 clades) and features of DNA methylation (high 6mA, ApT methylation, Pol II TSS association, and CpG methylation). High 6mA is defined as 6mA/A ratio higher than 0.1%. Star, triangle and square denote presence of AMT1, AMT6/7, and METTTL4 homologues in corresponding species. Solid circle, cross in circle, hollow circle, and hemi-solid circle denote presence, absence, no available data (N/A), variability/uncertainty in corresponding features of DNA methylation. See Supplementary Table S10 for details.

Article Snippet: After crosslink by 3% PFA, -6mA (Synaptic Systems, 202003, 1:2000) and its secondary antibody (goat anti-rabbit IgG (H+L), Alexa Fluor 488, Invitrogen, A-11008, 1:4000) were used.

Techniques: DNA Methylation Assay, Methylation, CpG Methylation Assay