Journal: Oxidative Medicine and Cellular Longevity

Article Title: circHtra1/miR-3960/GRB10 Axis Promotes Neuronal Loss and Immune Deficiency in Traumatic Brain Injury

doi: 10.1155/2022/3522492

Figure Lengend Snippet: IGF-1 reduces circHtra1 via ADAR1. (a) IGF-1 is immunoprecipitated with htra1 in HEK cells, and this effect is blocked by AG1024. (b) circHtra1 expression in neurons treated with KA, IGF-1, ADAR1, and their inhibitors ( t = 4.919, 3.162, and 8.503, respectively, df = 4). (c, d) Expression of ADAR1 in neurons treated with KA, IGF-1, ADAR1, and their inhibitors ( t = 3.123, 3.142, and 6.074, respectively, df = 4). (e, f) Expression of GRB10 in neurons treated with KA, IGF-1, ADAR1, and their inhibitors ( t = 11.242, 2.162, and 7.230, respectively, df = 4). ∗ , #, & P < 0.05.

Article Snippet: Goat β -actin antibody (ab8227) and rabbit anti-ADAR1 antibody (bs-2168R; Bioss, Woburn, MA, USA), rabbit polyclonal to GRB10 (ab125583; Abcam, Cambridge, UK), mouse monoclonal to Bcl-2 (ab692; Abcam), and rabbit monoclonal to anti-cleaved caspase-3 (EPR21032; Abcam) were used to evaluate apoptosis in mice with TBI.

Techniques: Immunoprecipitation, Expressing

Journal: bioRxiv

Article Title: Z-Form Stabilization By The Zα Domain Of Adar1p150 Has Subtle Effects On A-To-I Editing

doi: 10.1101/2025.06.02.657529

Figure Lengend Snippet: ( A ) The domain structure of the short (ADAR1p110) and long (ADAR1p150) isoforms of ADAR1 are shown. ADAR1p110 contains a deaminase domain which is responsible for ADAR1’s catalytic deamination activity, three double-stranded RNA binding domains (dsRBDs) which interact with the A-form structure of dsRNA, a Nuclear Localization Sequence (NLS) and a Zβ domain of unknown function. ADAR1p150 contains the same domain structure but has a ∼300 a.a. N-terminal extension which contains a Nuclear Export Sequence (NES) as well as a Zα domain. ( B ) ADAR1 deaminases adenosine to inosine in dsRNA, which replaces the amino group on the adenosine with a keto group and disrupts A-form helical structure at AU base pairs. ( C ) Cartoon model depicting editing of a dsRNA by ADAR1 and the different domains. ( D ) The Zα domain is able to stabilize the left-handed Z-conformation of dsDNA and dsRNA through key residues which stabilize the unique Z-form geometry.

Article Snippet: Western blots of ADAR1p150 were carried out identically as above using a rabbit anti-ADAR1p150 antibody (Cell Signaling, #32136).

Techniques: Activity Assay, RNA Binding Assay, Sequencing

Journal: bioRxiv

Article Title: Z-Form Stabilization By The Zα Domain Of Adar1p150 Has Subtle Effects On A-To-I Editing

doi: 10.1101/2025.06.02.657529

Figure Lengend Snippet: ( A ) Domain architectures and point mutant locations for the different re-integrated ADAR1p150 mutant constructs. Red stars indicated mutation sites. ( B ) Western blot (left) and quantification (right) showing doxycycline-inducible expression levels of the re-integrated ADAR1p150 mutants relative to re-integrated wild-type ADAR1p150. Quantification was from three replicates, all of which are shown in Supplemental Figures 5 and 6. ( C ) mRNA expression levels in Transcripts Per Million (TPM, on the y-axis ) of the re-integrated ADAR1p150 constructs for each cell line from RNA-seq data. For each transgene, the detected expression value is indicated for each sample. ( D ) Principal Component Analysis (PCA) of gene expression of the wild-type HEK293T cells, ADAR1 KO and ADAR1p150 KO HEK293T cells, and the ADAR1p150 re-integrated cell lines.

Article Snippet: Western blots of ADAR1p150 were carried out identically as above using a rabbit anti-ADAR1p150 antibody (Cell Signaling, #32136).

Techniques: Mutagenesis, Construct, Western Blot, Expressing, RNA Sequencing, Gene Expression

Journal: bioRxiv

Article Title: Z-Form Stabilization By The Zα Domain Of Adar1p150 Has Subtle Effects On A-To-I Editing

doi: 10.1101/2025.06.02.657529

Figure Lengend Snippet: ( A ) Illustration of how A-to-I editing events from aligned reads and unaligned reads are identified. Editing indices (the ratio of total A-to-G changes over the total number of adenosines) calculated for all REDIprotal sites ( B ) and Alu elements specifically ( C , AEI = Alu Editing Index) are shown for the different cell lines. Comparisons referenced to in the main text are marked and numbered. Each black dot represents a replicate and the red cross is the average of three replicates. ( D ) Similar to the editing index, the number of hyper-edited sites per million reads from originally unmapped reads is shown. ( E ) The editing index for ADAR1p150-specific REDIportal sites (by filtering out sites observed in the ADAR1p150 KO cell line) for all cell lines is shown. ( F ) The numbers of differentially edited sites for individual cell line pairs are compared y-axis as determined using DESeq2 . ( G ) Editing frequencies at the top 3 most differentially edited sites between wild-type and ADAR1p150 KO are shown.

Article Snippet: Western blots of ADAR1p150 were carried out identically as above using a rabbit anti-ADAR1p150 antibody (Cell Signaling, #32136).

Techniques:

Journal: bioRxiv

Article Title: Z-Form Stabilization By The Zα Domain Of Adar1p150 Has Subtle Effects On A-To-I Editing

doi: 10.1101/2025.06.02.657529

Figure Lengend Snippet: ( A ) Illustration of how editing clusters are identified, figure adapted from . ( B ) The cluster editing index, calculated by taking the number of A-to-G changes over the total number of adenosines per identified cluster, then averaged for all clusters. Comparisons which are mentioned in the main text are marked and numbered. Each black dot represents a replicate and the red cross is the average of three replicates. ( C ) The number of differentially edited clusters for individual cell line pairs are compared on the y-axis as determined using DESeq2 . ( D ) Zoomed in fragments of Alu foldbacks predicted by the CRSSANT software package that were also ADAR1p150-dependent (ie, they have differential editing when comparing the ADAR1p150* plus and minus doxycycline conditions). Editing sites were only plotted on the predicted dsRNAs if they had an average editing frequency of > 0.05 averaged over the 3 replicates.

Article Snippet: Western blots of ADAR1p150 were carried out identically as above using a rabbit anti-ADAR1p150 antibody (Cell Signaling, #32136).

Techniques: Software

Journal: bioRxiv

Article Title: Z-Form Stabilization By The Zα Domain Of Adar1p150 Has Subtle Effects On A-To-I Editing

doi: 10.1101/2025.06.02.657529

Figure Lengend Snippet: ( A ) Immunofluorescence images of the wild-type, ADAR1 KO, ADAR1p150 KO, re-integrated ADAR1p150 (ADAR1p150*), the N173S mutant (ADAR1p150 N173S *), and the N173A,Y177A (ADAR1p150 N173A,Y177A *) double mutant cell lines. The red signal is of an ADAR1p150-specific rabbit monoclonal antibody visualized using an Alexa Fluor 594 nm secondary antibody. The green signal is from G3BP1 which was endogenously tagged with EGFP, and the blue signal is DAPI. ( B ) The percentage of the red signal intensity (corresponding to the anti-ADAR1p150 antibody) in the cytoplasm versus the nucleus of the cell.

Article Snippet: Western blots of ADAR1p150 were carried out identically as above using a rabbit anti-ADAR1p150 antibody (Cell Signaling, #32136).

Techniques: Immunofluorescence, Mutagenesis

Journal: bioRxiv

Article Title: Z-Form Stabilization By The Zα Domain Of Adar1p150 Has Subtle Effects On A-To-I Editing

doi: 10.1101/2025.06.02.657529

Figure Lengend Snippet: ( A ) The structure of the Zα domain of ADAR1 bound to Z-RNA is shown (PDB: 2GXB) highlighting the water-mediated hydrogen bond between N173 and W195. ( B ) Domain structures of the ADAR1p150 N173S and ADAR1p150 N173S NES moved constructs. ( C ) Immunofluorescence images of the re-integrated wild-type ADAR1p150*, ADAR1p150 N173S , and ADAR1p150 N173S * NES moved cell lines. ( D ) percentage of Alexa Fluor 594 signal intensity (corresponding to anti-ADAR1p150 antibody staining) measured in the cytoplasm versus the nucleus of the cell for the ADAR1p150 N173S and ADAR1p150 N173S * NES moved cell lines.

Article Snippet: Western blots of ADAR1p150 were carried out identically as above using a rabbit anti-ADAR1p150 antibody (Cell Signaling, #32136).

Techniques: Construct, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Z-Form Stabilization By The Zα Domain Of Adar1p150 Has Subtle Effects On A-To-I Editing

doi: 10.1101/2025.06.02.657529

Figure Lengend Snippet: ( A ) Domain structures of ADAR1p150 and ZBP1 are shown. ( B ) A cartoon depiction of the two potential models showing the effect of the Zα domain on ADAR1 function. In model 1, the Zα domain augments A-to-I editing broadly in a sequence-independent manner. In model 2, the Zα domain of ADAR1 competes with ZBP1 for binding to Z-form substrates in an editing independent manner, thereby inhibiting cell death pathways.

Article Snippet: Western blots of ADAR1p150 were carried out identically as above using a rabbit anti-ADAR1p150 antibody (Cell Signaling, #32136).

Techniques: Sequencing, Binding Assay

Journal: NAR Cancer

Article Title: Over-expression of ADAR1 in mice does not initiate or accelerate cancer formation in vivo

doi: 10.1093/narcan/zcad023

Figure Lengend Snippet: Proposed model of ADAR1 action in cancer. ( A ) Proposed models of the action of ADAR1 in cancer. In the ‘ADAR1 as an oncogene’ model, elevated ADAR1 leads to increased A-to-I editing and this acts as a tumor initiating event or promotes tumor establishment and maintenance. In the alternate ‘ADAR1 as a passenger model’ ADAR1 is elevated as a result of changes in the tumor transcriptome and environment, leading to increased ADAR1 as a secondary consequence. ( B ) Schematic of the constructs used to overexpress murine Adar1 cDNA from the Rosa26 locus in mice.

Article Snippet: Membranes were blocked with 5% milk in Tris-buffered saline with tween (TBST) and incubated at 4°C overnight with rat monoclonal anti-mouse ADAR1 antibody (1:500 on primary tissue westerns or 1:1000 dilution for cell lines; clone RD4B11; inhouse hybridoma purified by Monash Antibody Technology Facility; also available from Merck Millipore as MABE1790) , Monoclonal Anti-FLAG M2-Peroxidase (HRP) antibody produced in mouse (1:2000 dilution; Sigma Aldrich, A8592), mouse anti-Actin (1:5000 dilution; Sigma Aldrich, MS-1295-P0).

Techniques: Construct

Journal: NAR Cancer

Article Title: Over-expression of ADAR1 in mice does not initiate or accelerate cancer formation in vivo

doi: 10.1093/narcan/zcad023

Figure Lengend Snippet: Overexpression of ADAR1 in vivo . ( A ) Schematic outline of in vivo validation experiment. ( B ) Representative flow cytometry histograms of peripheral blood leukocytes for GFP expression following 14 days in vivo tamoxifen treatment. ( C ) Western blot analysis of ADAR1 expression in the spleen (left panel) and liver (right panel) following 14 days in vivo tamoxifen treatment using anti-ADAR1 and anti-Flag antibodies. ( D ) Adar1 and Adarb1 expression as measured from RNA-seq of the liver following 14 days in vivo tamoxifen treatment ( n = 3–4 per genotype; note Adar1 -Za mutation samples were not sequenced).

Article Snippet: Membranes were blocked with 5% milk in Tris-buffered saline with tween (TBST) and incubated at 4°C overnight with rat monoclonal anti-mouse ADAR1 antibody (1:500 on primary tissue westerns or 1:1000 dilution for cell lines; clone RD4B11; inhouse hybridoma purified by Monash Antibody Technology Facility; also available from Merck Millipore as MABE1790) , Monoclonal Anti-FLAG M2-Peroxidase (HRP) antibody produced in mouse (1:2000 dilution; Sigma Aldrich, A8592), mouse anti-Actin (1:5000 dilution; Sigma Aldrich, MS-1295-P0).

Techniques: Over Expression, In Vivo, Biomarker Discovery, Flow Cytometry, Expressing, Western Blot, RNA Sequencing, Mutagenesis

Journal: NAR Cancer

Article Title: Over-expression of ADAR1 in mice does not initiate or accelerate cancer formation in vivo

doi: 10.1093/narcan/zcad023

Figure Lengend Snippet: Overexpression of ADAR1 in vivo increased A-to-I editing levels of cellular RNA. Gene expression analysis (MA plot, upper) and A-to-I editing levels (lower) of known individual sites following 14 days in vivo tamoxifen treatment from ( A ) Adar1 full length (FL); ( B ) Adar1p110; ( C ) Adar1p150; ( D ) Adar1E861A liver RNA-seq datasets compared to Ubc -CreER+ tamoxifen treated controls ( n = 3 per genotype). Red dots in upper panels represent interferon-stimulated genes. Blue dots in lower panels represent significantly different editing at individual sites between the genotypes (Jacusa statistic (likelihood ratio of two samples) >5). ( E ) The repeat editing index (AEI) from each genotype. ( F ) IGV screen shot of Azin1 editing at the recoding p.S367G site; quantitation and statistical analysis of the editing frequency at the recoding site and average number of reads per sample for the site (expressed as mean ± sem for each allele). ** P < 0.01, *** P < 0.001; Statistical comparisons using a two-way ANOVA with multiple comparisons correction using Prism software.

Article Snippet: Membranes were blocked with 5% milk in Tris-buffered saline with tween (TBST) and incubated at 4°C overnight with rat monoclonal anti-mouse ADAR1 antibody (1:500 on primary tissue westerns or 1:1000 dilution for cell lines; clone RD4B11; inhouse hybridoma purified by Monash Antibody Technology Facility; also available from Merck Millipore as MABE1790) , Monoclonal Anti-FLAG M2-Peroxidase (HRP) antibody produced in mouse (1:2000 dilution; Sigma Aldrich, A8592), mouse anti-Actin (1:5000 dilution; Sigma Aldrich, MS-1295-P0).

Techniques: Over Expression, In Vivo, Gene Expression, RNA Sequencing, Quantitation Assay, Software

Journal: NAR Cancer

Article Title: Over-expression of ADAR1 in mice does not initiate or accelerate cancer formation in vivo

doi: 10.1093/narcan/zcad023

Figure Lengend Snippet: Long-term in vivo overexpression of ADAR1 is well tolerated. ( A ) Schematic outline of in vivo experiment. ( B ) Kaplan–Meier survival plot of each genotype. Numbers as indicated in the inset, no significant difference in survival. Peripheral blood ( C ) leukocyte numbers, ( D ) red blood cell numbers and ( E ) platelet counts for each genotype; number per genotype indicated in panel C noting that the range indicates the minimum and maximum at any given time point per genotype. Due to restrictions during the pandemic the time points have been assigned as 0, 14/28 days, 84 days, 175 days and >580 days and the time points grouped to the closest of these for graphing. ( F ) GFP levels in the total leukocyte population. ( G ) Representative flow cytometry plots showing GFP levels in each genotype at the indicated time points. If no statistical significance indicated then no significant difference.

Article Snippet: Membranes were blocked with 5% milk in Tris-buffered saline with tween (TBST) and incubated at 4°C overnight with rat monoclonal anti-mouse ADAR1 antibody (1:500 on primary tissue westerns or 1:1000 dilution for cell lines; clone RD4B11; inhouse hybridoma purified by Monash Antibody Technology Facility; also available from Merck Millipore as MABE1790) , Monoclonal Anti-FLAG M2-Peroxidase (HRP) antibody produced in mouse (1:2000 dilution; Sigma Aldrich, A8592), mouse anti-Actin (1:5000 dilution; Sigma Aldrich, MS-1295-P0).

Techniques: In Vivo, Over Expression, Flow Cytometry

Journal: NAR Cancer

Article Title: Over-expression of ADAR1 in mice does not initiate or accelerate cancer formation in vivo

doi: 10.1093/narcan/zcad023

Figure Lengend Snippet: Modest changes in hematopoiesis with overexpression of ADAR1. ( A ) The percentage contribution of peripheral blood GFP+ cells to each indicated lineage across each genotype. ( B ) Total bone marrow cellularity per femur. ( C ) The percentage contribution of GFP+ cells in the bone marrow to each indicated population. ( D ) Outline of hematopoiesis and the relationship between populations assessed. ( E ) Representative flow cytometry plots used to define GFP positive and negative fractions and ( F ) assess the hematopoietic stem and progenitor compartment. ( G ) The percentage contribution of GFP+ cells in the bone marrow to the lineage-cKit + Sca1+ (LKS+) population and the long-term and short-term hematopoietic stem cell populations (contained within the LKS+ fraction). ( H ) The percentage contribution of GFP+ cells in the bone marrow to the lineage-cKit+ Sca1– (LKS–) population and the megakaryocyte progenitors (MkP), granulocyte macrophage progenitors (GMP), pre-GM, pre-Megakaryocyte erythroid progenitors (preMegE), pre colony forming unit erythroid (preCFU-E) and CFU-E populations (contained within the LKS- fraction). ( I ) Total cellularity of the spleen and contribution of the GFP+ cells to the indicated cell populations. ( J ) Total thymus cellularity and contribution of the GFP+ cells to the indicated cell populations. Each circle indicates an individual animal; * P < 0.05, ** P < 0.01, *** P < 0.001; statistical comparisons using a two-way ANOVA with multiple comparisons correction using Prism software.

Article Snippet: Membranes were blocked with 5% milk in Tris-buffered saline with tween (TBST) and incubated at 4°C overnight with rat monoclonal anti-mouse ADAR1 antibody (1:500 on primary tissue westerns or 1:1000 dilution for cell lines; clone RD4B11; inhouse hybridoma purified by Monash Antibody Technology Facility; also available from Merck Millipore as MABE1790) , Monoclonal Anti-FLAG M2-Peroxidase (HRP) antibody produced in mouse (1:2000 dilution; Sigma Aldrich, A8592), mouse anti-Actin (1:5000 dilution; Sigma Aldrich, MS-1295-P0).

Techniques: Over Expression, Flow Cytometry, Software

Journal: NAR Cancer

Article Title: Over-expression of ADAR1 in mice does not initiate or accelerate cancer formation in vivo

doi: 10.1093/narcan/zcad023

Figure Lengend Snippet: Adar1 expression and editing activity increases during cellular immortalization. ( A ) Schematic outline of in vitro experiment. Long bone osteoblasts were isolated from R26 -CreER T2 Trp53 fl/fl mice and cultured with tamoxifen to induce deletion of p53. Cells were collected at day 7, 14 and 21 for analysis. Osteoblasts were isolated from three animals and cultured separately (biological replicates). ( B ) Expression (counts per million) of Adar1 and Adarb1 at day 7, 14 and 21 as determined by RNA-seq at each time point. ( C ) MA plot of gene expression comparing day 7 (p53 still expressed) and day 21 (p53 deficient) with significantly different genes indicated in blue and interferon stimulated genes (ISGs) indicated in red. ( D ) IGV screen shot of Azin1 (upper) and Cdk13 (lower) editing at the indicated recoding sites; quantitation of the editing frequency at each site (expressed as mean ± sem for each allele). The Alu/repeat editing index (AEI) from each timepoint derived from the RNA-seq. ( E ) A-to-I editing levels of known individual sites comparing editing levels at day 21 (y axis) to day 7 (x axis) following tamoxifen treatment. Blue dots represent significantly different editing at individual sites between the genotypes (Jacusa statistic (likelihood ratio of two samples) >5). ( F ) The repeat editing index (AEI) at each time point calculated from the RNA-seq dataset. *** P < 0.001; Statistical comparisons using a two-way ANOVA with multiple comparisons correction.

Article Snippet: Membranes were blocked with 5% milk in Tris-buffered saline with tween (TBST) and incubated at 4°C overnight with rat monoclonal anti-mouse ADAR1 antibody (1:500 on primary tissue westerns or 1:1000 dilution for cell lines; clone RD4B11; inhouse hybridoma purified by Monash Antibody Technology Facility; also available from Merck Millipore as MABE1790) , Monoclonal Anti-FLAG M2-Peroxidase (HRP) antibody produced in mouse (1:2000 dilution; Sigma Aldrich, A8592), mouse anti-Actin (1:5000 dilution; Sigma Aldrich, MS-1295-P0).

Techniques: Expressing, Activity Assay, In Vitro, Isolation, Cell Culture, RNA Sequencing, Gene Expression, Quantitation Assay, Derivative Assay

Journal: NAR Cancer

Article Title: Over-expression of ADAR1 in mice does not initiate or accelerate cancer formation in vivo

doi: 10.1093/narcan/zcad023

Figure Lengend Snippet: Overexpression of ADAR1 does not accelerate or modify osteosarcoma behavior in vivo . ( A ) Schematic outline of in vivo osteosarcoma model. ( B ) Kaplan–Meier survival plot of each genotype. Numbers as indicated in the inset, no significant difference in survival. Number of animals per genotype indicated in inset. ( C ) Median days of age of each genotype, same cohort as represented in the KM plot; assessed by two-way ANOVA with multiple comparison correction. ( D ) Analysis of primary tumor location and metastatic spread in each genotype. ( E ) Representative histology of primary and metastatic lesions from each indicated genotype. The osteosarcoma model generates a fibroblastic osteosarcoma. ( F ) Western blot of ADAR1 (anti-ADAR1 antibody) in whole tumor pieces derived from the indicated genotypes.

Article Snippet: Membranes were blocked with 5% milk in Tris-buffered saline with tween (TBST) and incubated at 4°C overnight with rat monoclonal anti-mouse ADAR1 antibody (1:500 on primary tissue westerns or 1:1000 dilution for cell lines; clone RD4B11; inhouse hybridoma purified by Monash Antibody Technology Facility; also available from Merck Millipore as MABE1790) , Monoclonal Anti-FLAG M2-Peroxidase (HRP) antibody produced in mouse (1:2000 dilution; Sigma Aldrich, A8592), mouse anti-Actin (1:5000 dilution; Sigma Aldrich, MS-1295-P0).

Techniques: Over Expression, In Vivo, Comparison, Western Blot, Derivative Assay