human adar1 p150  (Addgene inc)

Journal: Journal of Translational Medicine

Article Title: An RNA editing fingerprint of cancer stem cell reprogramming

doi: 10.1186/s12967-014-0370-3

Figure Lengend Snippet: Identification of an RNA editing fingerprint of malignant progenitor reprogramming, and stable ADAR1 overexpression in K562 cells. (A) LSC purification strategy for detection of CSC-associated RNA recoding. (B-D) RNA-sequencing analysis of FACS-purified CP and BC CML LSC showing A-to-G RNA editing changes in MDM2, AZIN1 and APOBEC3D (n = 8 per group). (E) Lentiviral construct expressing human ADAR1 or a catalytically inactive form (ADAR1m). (F-H) qRT-PCR analysis of cDNA prepared from K562 lines using primers detecting ADAR1 lentivirus (F) and total human ADAR1 (G,H) showing K562 leukemia cells stably transduced with active ADAR1 or inactive ADAR1m express high levels of ADAR1 transcripts compared with vector open reading frame (ORF) control backbone. *p < 0.05 by unpaired, two-tailed Student’s t -test.

Article Snippet: We have previously characterized lentiviral vectors (Thermo Scientific) for overexpression of human ADAR1 p150-IRES-GFP [ ].

Techniques: Over Expression, Purification, RNA Sequencing, Construct, Expressing, Quantitative RT-PCR, Stable Transfection, Transduction, Plasmid Preparation, Control, Two Tailed Test

Journal: Journal of Translational Medicine

Article Title: An RNA editing fingerprint of cancer stem cell reprogramming

doi: 10.1186/s12967-014-0370-3

Figure Lengend Snippet: Validation and quantification of RNA editing activity in primary bone marrow-derived hematopoietic stem and progenitor cells transduced with lentiviral-ADAR1. CD34-selected cells from normal bone marrow (BM) samples (n = 3, average donor age = 64.3 ± 2.9 years old) were transduced with lentiviral (lenti)-ADAR1 or vector (ORF) control. After 4 days of culture, cells were lysed and processed for qRT-PCR and RESSq-PCR analysis. (A,B) Relative expression of lentivirus-derived (a) and total (b) ADAR1 levels in transduced BM samples (n = 3) showing increased human ADAR1 expression in ADAR1-transduced samples, with higher levels of total ADAR1 overexpression achieved in samples BM-410 and BM-416. (C,D) Representative Sanger sequencing analysis of high-fidelity PCR products amplified with primers flanking the APOBEC3D editing site showing increased G(I) peak in lenti-ADAR1 transduced cells that displayed robust ADAR1 expression (BM-410, C). (E,F) Quantification of sequencing peak height ratios and corresponding RESSq-PCR analysis in lenti-ORF and lenti-ADAR1 transduced BM samples.

Article Snippet: We have previously characterized lentiviral vectors (Thermo Scientific) for overexpression of human ADAR1 p150-IRES-GFP [ ].

Techniques: Biomarker Discovery, Activity Assay, Derivative Assay, Transduction, Plasmid Preparation, Control, Quantitative RT-PCR, Expressing, Over Expression, Sequencing, Amplification

Journal: Journal of Translational Medicine

Article Title: An RNA editing fingerprint of cancer stem cell reprogramming

doi: 10.1186/s12967-014-0370-3

Figure Lengend Snippet: In vitro humanized stromal co-culture model and RESSq-PCR analysis of primary CP CML cells transduced with lentiviral-ADAR1. (A) Schematic diagram of humanized bone marrow stromal co-culture assay. CD34-selected hematopoietic stem and progenitor cells (HSPC) isolated from patients with CP CML were transduced with lenti-ADAR1 or ORF control. After 3 days of culture, cells were transferred to SL/M2 mouse bone marrow stromal monolayers for co-culture and subsequent RESSq-PCR analysis. (B,C) Increased total ADAR1 (B) and lenti-ADAR1 (C) expression in transduced CP CML samples (n = 3). (D) RESSq-PCR analysis showing increased APOBEC3D RNA editing in lenti-ADAR1 transduced cells from patients with CP CML that harbored high ADAR1 expression after transduction. Horizontal dashed lines represent comparative RNA editing activity in K562-ADAR1 and K562-ORF cells.

Article Snippet: We have previously characterized lentiviral vectors (Thermo Scientific) for overexpression of human ADAR1 p150-IRES-GFP [ ].

Techniques: In Vitro, Co-Culture Assay, Transduction, Co-culture Assay, Isolation, Control, Expressing, Activity Assay

Journal: bioRxiv

Article Title: Re: Concerns Regarding the Validation of ZYS-1 as a Bona Fide ADAR1 Inhibitor

doi: 10.1101/2025.03.07.641892

Figure Lengend Snippet: (A) The sequences and secondary structures of dsRNAs used in our assays. HT-dsRNA, GLI-dsRNA-1, GLI-dsRNA-2 are the ADAR1 editing substrates, containing adenosine editing sites. GLI-dsRNA-Product is the ADAR1 editing product, containing inosine at the editing site. (B) Responses of adenosine deaminase activity (ADA) assay kit to inosine, ADAR1 editing substrate GLI-dsRNA-2 and ADAR1 editing product GLI-dsRNA-Product. (C) Representative gel images showing the RNA editing activity of human ADAR1 in the presence or absence of the ZYS-1 compound. Reactions were performed with 50 nM HT-dsRNA or GLI-dsRNA-1 and 150 nM ADAR1, with ZYS-1 concentrations as indicated. The observed product results from ADAR1-mediated RNA editing, followed by cleavage with EcEndoV, which specifically targets inosine-containing RNA. (D) Quantification of normalized RNA editing activity from (C). Each experiment was independently repeated at least three times.

Article Snippet: The gene of human ADAR1 was obtained from Addgene (plasmid #117927).

Techniques: Activity Assay

Journal: bioRxiv

Article Title: All-trans retinoic acid-mediated ADAR1 degradation synergizes with PD-1 blockade to suppress pancreatic cancer

doi: 10.1101/2024.10.20.619300

Figure Lengend Snippet: (A) Western blots showing the protein levels of ADAR1 and PD-L1 in Capan1, HPAC, BT549 and MB-231 cells treated with the indicated concentrations of ATRA for 24 h. PDAC, pancreatic ductal adenocarcinoma; TNBC, triple-negative breast cancer. (B) Western blots showing the protein levels of Adar1 and Pd-l1 in Panc02 and 4T1 cells treated with the indicated doses of ATRA for 24 h. (C) Western blots showing exogenous MYC-GFP-tagged ADAR1 protein expression in HPAC or MB-231 cells treated with ATRA for 24 h. (D) Western blots showing exogenous FLAG-tagged ADAR1 protein expression in BxPC3 cells treated with ATRA for 24 h. (E) Upper, Western blots showing the protein levels of ADAR1 in HPAC cells treated with ATRA or MG132 alone or in combination for the indicated time. Lower, Quantification of relative ADAR1 expression. (F) Co-immunoprecipitation (Co- IP) blots showing polyubiquitination (Ub) and K48-linked ubiquitination (Ub-K48) of ADAR1 in BxPC3 and Capan1 cells exogenously expressing MYC-GFP-tagged ADAR1 and treated with ATRA. (G) Co-IP blots showing polyubiquitination and Ub-K48 of ADAR1 in HPAC cells treated with ATRA or MG132.

Article Snippet: Human ADAR1 (p150 isoform) antibody was purchased from Bethyl Laboratories, Inc. Human ADAR1 (p110 isoform) antibody and vinculin antibody were purchased from Proteintech.

Techniques: Western Blot, Expressing, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: bioRxiv

Article Title: All-trans retinoic acid-mediated ADAR1 degradation synergizes with PD-1 blockade to suppress pancreatic cancer

doi: 10.1101/2024.10.20.619300

Figure Lengend Snippet: (A) Schematic representation of the treatment schedule in the Panc02 mouse tumor model. Panc02 tumor-bearing mice were given ATRA or placebo pellets (10 mg per mouse, subcutaneously [s.c.], 21-day release). Administration of anti-PD-1 antibody or IgG control (100 μg per mouse, intraperitoneally [i.p.], 3 times weekly) was started along with ATRA or placebo pellets. Tumor growth, body weight, and mouse survival were measured. (B) Left, representative images showing Panc02 tumors harvested from mice that received the indicated treatments. Scale bar, 1.2 cm. Right, tumor weight. Data represent mean ± SD. (C) Tumor growth curves of individual mice injected with Panc02 tumor cells and subjected to the indicated treatments. (D) Mean tumor growth in mice injected with Panc02 tumor cells and subjected to the indicated treatments. Each dot represents the mean tumor size from 9 mice in each treatment group; whiskers represent SD. (E) Survival curves of mice that received the indicated treatments (9 mice/group). (F) Survival curves of KPC mice that received the indicated treatments (3 mice/group). (G) Western blots showing Adar1 and Pd-l1 protein expression levels in Panc02 cells from mice treated with the indicated regimens. (H) Body weight of tumor-bearing mice treated with the indicated treatments. Data are shown as mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant.

Article Snippet: Human ADAR1 (p150 isoform) antibody was purchased from Bethyl Laboratories, Inc. Human ADAR1 (p110 isoform) antibody and vinculin antibody were purchased from Proteintech.

Techniques: Control, Injection, Western Blot, Expressing

Journal: bioRxiv

Article Title: All-trans retinoic acid-mediated ADAR1 degradation synergizes with PD-1 blockade to suppress pancreatic cancer

doi: 10.1101/2024.10.20.619300

Figure Lengend Snippet: (A) Heatmap showing differential marker expression in CD45+ tumor-infiltrating lymphocyte (TIL) clusters identified by analysis of time-of-flight mass cytometry data with viSNE and FlowSOM. DC, dendritic cell; MDSC, myeloid-derived suppressor cell; Treg, regulatory T cell; NK, natural killer cell. (B) Annotation of TIL populations based on differential marker expression as shown in the heatmap (A) and Figure S8. TAM, tumor-associated macrophage. (C) Cellular distribution and clustering as defined by tSNE1 and tSNE2, colored by cell phenotype, for Panc02 tumors subjected to the indicated treatments. Data show all normalized viable single cells; analysis was conducted using viSNE and FlowSOM. (D) Percentage of CD8+ T cells among total CD45+ TILs under the indicated treatments. (E) T-cell cytotoxicity (CCK-8) showing effects of the indicated treatments on pancreatic cancer cell viability. Target Panc28 cancer cells were engineered to express Fcγ receptor 2A fused with the luciferase Luc2 at the C- terminus (Panc28-RL2), and then incubated with CD8+ T cells and the CD3 antibody OKT3 for 3 days. Data represent mean ± SD. P values were calculated by Student t test. (F) T-cell cytotoxicity (CCK-8) assay showing effects of ATRA on pancreatic cancer cell viability. Panc28-RL2 cells overexpressing MYC-GFP-ADAR1 or MYC-GFP (control) were incubated with CD8+ T cells and the CD3 antibody OKT3 for 3 days. Data represent mean ± SD. P values calculated by Student t test.

Article Snippet: Human ADAR1 (p150 isoform) antibody was purchased from Bethyl Laboratories, Inc. Human ADAR1 (p110 isoform) antibody and vinculin antibody were purchased from Proteintech.

Techniques: Marker, Expressing, Mass Cytometry, Derivative Assay, CCK-8 Assay, Luciferase, Incubation, Control

Journal: bioRxiv

Article Title: All-trans retinoic acid-mediated ADAR1 degradation synergizes with PD-1 blockade to suppress pancreatic cancer

doi: 10.1101/2024.10.20.619300

Figure Lengend Snippet: (A) ADAR gene expression profiling by GEPIA2. Red dots: tumor tissues; green dots: normal tissues. The bars represent the median expression of certain tumor type or normal tissue. BRCA, invasive breast carcinoma; ESCA, esophageal carcinoma; HNSC, head and neck squamous cell carcinoma; LGG, lower-grade brain glioma; LIHC, liver hepatocellular carcinoma; PAAD, pancreatic adenocarcinoma; SKCM, skin cutaneous melanoma; STAD, stomach adenocarcinoma; TGCT, testicular germ cell tumor. (B) Kaplan-Meier overall survival curves of 135 pancreatic cancer patients in the cohort with high and low ADAR1 expression determined by immunohistochemical staining. (C) Representative images of the immunohistochemical staining of ADAR1 in tumor tissue from patients in the MDA cohort. Scale bars, 50 μm. (D) Survival curves of breast cancer patients from 2 independent cohorts by ADAR1 expression level. (i) Liu et al. , 2014; (ii) TCGA. (E) Representative images of the immunohistochemical staining of ADAR1 and PD-L1 in tumor tissue from pancreatic cancer patients before treatment with ATRA and nivolumab. Scale bars, 100 μm. (F) Kaplan-Meier overall survival curves for 9 pancreatic cancer patients treated with ATRA 45 mg/m 2 /day plus nivolumab or ATRA ≥60 mg/m 2 /day plus nivolumab. (G) Schematic illustration of the mechanism of switching “cold” (immune-excluded) tumors to “hot” (immune-infiltrated) tumors by using ATRA and anti-PD-1 antibody.

Article Snippet: Human ADAR1 (p150 isoform) antibody was purchased from Bethyl Laboratories, Inc. Human ADAR1 (p110 isoform) antibody and vinculin antibody were purchased from Proteintech.

Techniques: Expressing, Immunohistochemical staining, Staining