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: Genome-wide analysis of consistently RNA edited sites in human blood reveals interactions with mRNA processing genes and suggests correlations with cell types and biological variables

doi: 10.1101/254045

Figure Lengend Snippet: Association between gene expression and CES total editing rate We analyzed association between CES total editing rate and gene expression for 14,961 human genes. (a) Gene set enrichment analysis by hypergeometric test on GO-BP categories and REACTOME pathways revealed that associated genes are mainly involved in immune system response mediated by interferon I and alpha / beta. (b) When we analyze distribution of CES total editing rate and ADAR gene expression, ADAR expression levels explains ∼ 13% of observed variability. No significant effect is observed for ADARB1 expression. ADAR and ADARB1 expression levels are reported as residuals of ridge regression with technical covariates (see description of data in methods section). The graphs report adjusted p-value and R2 value from robust regression analysis. (c) The 1,122 genes associated to CES total editing rate after removing ADAR expression effect were enriched for genes mainly involved in ribonucleoprotein and RNA processing.

Article Snippet: During the immunoblot step the following primary antibodies were used 1h at RT: mouse anti-ADAR1 (Santa Cruz, cod. sc-73408) 1:300 in 5% non-fat dry milk in TBST 0,1%; mouse anti-IFI16 (Abcam, cod.

Techniques: Expressing

Journal: bioRxiv

Article Title: Genome-wide analysis of consistently RNA edited sites in human blood reveals interactions with mRNA processing genes and suggests correlations with cell types and biological variables

doi: 10.1101/254045

Figure Lengend Snippet: Genes associated with CES total editing rate are enriched for ADAR interactors (a) Reconstructed PPI network including ADARs and proteins encoded by best genes significantly associated with global editing levels (FDR < 0.01). Among these proteins, we observed 285 potential ADARs interactors, including 9 direct partners of ADARs proteins. (b) Boxplot of number of ADARs interacting genes observed in 1M random simulations. The observed number of interactions (285) resulted in empirical p-value < 1e-6. (c) ADARs interactors are strongly enriched for RNA binding proteins in GO-MF categories. (d) Distribution of degree and betweenness centrality values among network nodes are represented by violin plots. ADAR1 protein has a major role (higher values) among ADAR proteins. Among ADARs direct partners, ELAVL1, RPA1 and IFI16 showed high values of degree and betweenness centrality, suggesting a central role in the network. (e) ADAR1 interaction with RPA70 (coded by RPA1) and IFI16 determined by co-immunoprecipitation. After immunoprecipitation with ADAR1 antibody, western blot for IFI16 and RPA70 are reported. For a better discrimination two times of exposure are reported in the figure.

Article Snippet: During the immunoblot step the following primary antibodies were used 1h at RT: mouse anti-ADAR1 (Santa Cruz, cod. sc-73408) 1:300 in 5% non-fat dry milk in TBST 0,1%; mouse anti-IFI16 (Abcam, cod.

Techniques: RNA Binding Assay, Immunoprecipitation, Western Blot

Journal: bioRxiv

Article Title: Genome-wide analysis of consistently RNA edited sites in human blood reveals interactions with mRNA processing genes and suggests correlations with cell types and biological variables

doi: 10.1101/254045

Figure Lengend Snippet: Impact of cell composition on CES total editing rate and ADAR / ADARB1 expression Our analysis revealed strong associations with CES total editing rate for 4 cell type variables (a), representing proportion of neutrophils, monocytes, dendritic cells (DC) and T helper (Th). Specific cell variables resulted significantly associated also to ADAR (b) and ADARB1 (c) expression. Significance level (p) and correlation coefficient (r) are reported in each plot based on Pearson’s product-moment. Only non-zero observations are plotted.

Article Snippet: During the immunoblot step the following primary antibodies were used 1h at RT: mouse anti-ADAR1 (Santa Cruz, cod. sc-73408) 1:300 in 5% non-fat dry milk in TBST 0,1%; mouse anti-IFI16 (Abcam, cod.

Techniques: Expressing

Journal: bioRxiv

Article Title: Genome-wide analysis of consistently RNA edited sites in human blood reveals interactions with mRNA processing genes and suggests correlations with cell types and biological variables

doi: 10.1101/254045

Figure Lengend Snippet: Impact of biological / pharmacological factors on CES total editing rate and ADAR / ADARB1 expression Our analysis revealed significant associations with CES total editing rate for blood pressure medication, BMI current, Age and Sex (a). Specific biological variables resulted significantly associated also to ADAR (b) and ADARB1 (c) expression. Significance level of association after correction for cell composition is reported (p (cell)) is reported in each plot based on Mann-Whitney-Wilcoxon or Pearson’s product-moment correlation test for binary and continuous variables, respectively. For continuous variables the Pearson correlation coefficient (r) is also reported.

Article Snippet: During the immunoblot step the following primary antibodies were used 1h at RT: mouse anti-ADAR1 (Santa Cruz, cod. sc-73408) 1:300 in 5% non-fat dry milk in TBST 0,1%; mouse anti-IFI16 (Abcam, cod.

Techniques: Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: Genome-wide analysis of consistently RNA edited sites in human blood reveals interactions with mRNA processing genes and suggests correlations with cell types and biological variables

doi: 10.1101/254045

Figure Lengend Snippet: Impact of cell composition, biological and pharmacological factors on PCs of editing levels The heathmap represents strength of association between the first 5 principal components of CESs (PCs) and ADAR / ADARB1 expression (upper panel), 7 cell composition variables (middle panel) and 11 biological / pharmacological variables (lower panel). Only factors showing significant association with at least one of the first 5 PCs are represented. Significant p values (< 0.05) are colored in yellow-red scale, while p value > 0.05 are represented in grey scale. Age, BMI, blood pressure medications, smoke and alcohol all associate with PC1. Also time of blood draw seems to have a small, but consistent effect, on different PCs. For each PC, variance explained is represented by the bar plot in the upper side.

Article Snippet: During the immunoblot step the following primary antibodies were used 1h at RT: mouse anti-ADAR1 (Santa Cruz, cod. sc-73408) 1:300 in 5% non-fat dry milk in TBST 0,1%; mouse anti-IFI16 (Abcam, cod.

Techniques: Expressing

Journal: bioRxiv

Article Title: Genome-wide analysis of consistently RNA edited sites in human blood reveals interactions with mRNA processing genes and suggests correlations with cell types and biological variables

doi: 10.1101/254045

Figure Lengend Snippet: Association study for SNPs and CES total editing rate (a) Manhattan plot representing the association between 573,801 SNPs and CES total editing rate, where black line represents threshold for the top 100 SNPs (p value ∼ 10e-4). (b) Detailed view of genotyped SNPs located in the region at chromosome 7 that showed significant association with CES total editing rate. Known GWAS associations for human phenotypes from GRASP database are reported in the lower panel. (c) The top associated SNP (rs856554) showed a significant effect on global editing level, while no significant correlation was observed with ADAR and ADARB1 expression. (d) Real-time expression analysis of ADAR and ADARB1 mRNA after B-EBV transfection of LOC730338. Not transfected cells were used as control samples. Data are reported as 2 -ΔΔ ct (expression level of control sample is equal to 1) and represent mean values and standard errors obtained from at least 3 independent evaluations. Unpaired t test was used for statistical analysis (*p< 0.05).

Article Snippet: During the immunoblot step the following primary antibodies were used 1h at RT: mouse anti-ADAR1 (Santa Cruz, cod. sc-73408) 1:300 in 5% non-fat dry milk in TBST 0,1%; mouse anti-IFI16 (Abcam, cod.

Techniques: Expressing, Transfection

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