Journal: Cancer cell

Article Title: Loss of SYNCRIP unleashes APOBEC-driven mutagenesis, tumor heterogeneity, and AR-targeted therapy resistance in prostate cancer

doi: 10.1016/j.ccell.2023.06.010

Figure Lengend Snippet:

Article Snippet: Rabbit anti-APOBEC1 (Millipore, ABE438), Rabbit anti-APOBEC3A (Invitrogen, Cat# PA5–78800), Rabbit anti-APOBEC3B mAb (CST, #41494), Rabbit anti-APOBEC3C (Proteintech, 10591–1-AP), Rabbit anti-FOXA1 (CST, #53528), Rabbit anti-cyclophilin B (CST, #43603).

Techniques: Magnetic Beads, Recombinant, Infection, Western Blot, SYBR Green Assay, Bicinchoninic Acid Protein Assay, Transfection, Purification, Gel Extraction, Cell Viability Assay, Single Cell Gel Electrophoresis, Software

Journal: Cell reports

Article Title: Inflammation Driven Deaminase Deregulation Fuels Human Pre-leukemia Stem Cell Evolution

doi: 10.1016/j.celrep.2020.108670

Figure Lengend Snippet: A. Sample Distribution in this study. Samples were distributed among Polycythemia Vera (PV, n=5), Essential Thrombocythemia (ET, n=4), Myelofibrosis (MF, n=28), Chronic Myeloid Leukemia (CML, n=3) and non-MPN control individuals (n=4, including 3 healthy volunteers and 1 CLL with CALR SNP). In parallel, whole-genome sequencing of 43 peripheral blood samples of a sample distribution of PV (n=6), ET (n=4), MF (n=26), CML (n=3) and non-MPN control individuals (n=4, including 1 CLL with CALR SNP). The somatic mutations were obtained from MPN patient samples (n=37) and non-MPN controls (healthy controls n=3 and CLL with CALR SNP n=1) with matching saliva (30X coverage) and peripheral blood (n=41, shown in solid black). Whole-transcriptomic sequencing (RNA-seq) was performed on 78 samples distributed as follows: PV (n=6), ET (n=2), MF (n=29), CML (n=5), AML (n=12), and non-MPN control individuals (n=24). These samples can further be broken down based on tissue of collection (peripheral blood or bone marrow) and cell types (stem cells and progenitor). In summary, from 54 subjects and 24 non-MPN controls, 113 samples were represented in the RNA sequencing analysis. B. Mutational burden of single point mutations (log-scaled). Each dot represents the number of substitutions per megabase in an individual MPN sample. Red lines reflect median numbers. Mutational profiles of substitutions are shown using six subtypes: C>A, C>G, C>T, T>A, T>C, T>G. Underneath each subtype are 16 bars reflecting the sequence contexts determined by the four possible bases 5’ and 3’ each mutated base. Average contributions of the two clock-like signatures across PCAWG MPN and MCCWG MPN samples are shown in different colors. C. Mutations in 69 MPN-associated genes (Grinfeld et al., 2018) in peripheral blood divided by MPN disease stage. Clinical-grade confirmation of JAK2 V617F mutation was marked as light yellow in MPN patients. MPN disease stage depicted in colored bar at the bottom of the figure. *, patient deceased since sample collection; +, patient has another malignancy; &, patient progressed after sample collection, and &&, patient progressed to AML after sample collection. D. A boxplot depicting the number of somatic mutations in peripheral blood or saliva based on transitions (Ti) or transversions (Tv). Both somatic and germline variants were included. E. A boxplot depicting the expression levels of APOBEC3 in ABM, YBM, intermediate-risk myelofibrosis (Int-MF), high-risk myelofibrosis (HR-MF) and sAML stem cell populations using normalized RNA-Seq. APOBEC3C expression was illustrated for each stem cell sample compared with ABM normal controls. (p < 0.05 =*). F. Comparison of the HSC percentage in MPN samples by flow cytometry (CML n=4, PV n=3, ET n=2, MF n=23 and AML n=3). G. A representative brightfield microscopic image of cord blood CD34+ cells lentivirally transduced with APOBEC3C compared with a lentiviral backbone control (left). H. Flow cytometry analysis of cord blood CD34+ cells 48 hours after lentiviral transduction. Error bars show SEM and significance determined by 2way ANOVA.

Article Snippet: Membranes were incubated with 5% BSA in TBS-T for blocking and probed with primary and secondary antibodies diluted in 5% BSA in TBS-T. APOBEC3C lentiviral Vectors Lentiviral human wild-type APOBEC3C (pCDH-EF1-T2A-copGFP) was cloned by Eton Biosciences.

Techniques: Sequencing, RNA Sequencing Assay, Mutagenesis, Expressing, Flow Cytometry, Transduction

Journal: Cell reports

Article Title: Inflammation Driven Deaminase Deregulation Fuels Human Pre-leukemia Stem Cell Evolution

doi: 10.1016/j.celrep.2020.108670

Figure Lengend Snippet: A. Heatmap of RNA-Seq expression of splicing isoforms for the top 1% of genes ranked by variance. Annotation for each sample is presented as a stack of colored bars representing phenotype, cell type, source tissue, mutation status, and the treatment type (for MF samples only). Samples without a known JAK2 V617F mutation status are colored in gray. B. A boxplot representing the internally normalized expression of IL6ST isoforms (ENST00000381298 and ENST00000503773 in stem cells) and (ENST00000381298 and ENST00000336909 in the progenitors) in each MPN phenotype. Black dots represent expression values in lowest 2.5% or highest 97.5% of the distribution. C. Ratio of ADAR1 isoforms (p150/p110) in each MPN disease type using RNA-Seq expression data from stem cells and progenitors. D. Signaling Pathway Impact Analysis (SPIA) was performed for ET, PV, MF and AML compared to ABM progenitors. Listed are the top 5 activated pathways based on the NDE (number of differentially expressed genes per pathway)/pSize (number of genes in the pathway) in percent. E. SPIA in cord blood lentivirally transduced with ADAR1 WT (top) or RNA deaminase deficient mutant ADAR1E912A (bottom) compared to pCDH backbone controls (n=3). Listed are the top 6 activated pathways based on the NDE/pSize in percent. F. Correlation of normalized and Log2-transformed counts per million (CPM) data for APOBEC3C with ADAR1 p150 isoform in stem cells (top) and progenitors (bottom). Points are colored by phenotype. The MF risk-group is indicated by point shape with open shapes representing deceased patients. G. Western blot probed for ADAR1 p150 after co-immunoprecipitation with ADAR1 and APOBEC3C-FLAG. H. Colocalization of APOBEC3C and ADAR1 in TF1a cells. Immunofluorescence of anti-APOBEC3C (green) and anti-ADAR1 p150-specific (red) antibodies in TF1a shADAR1 and TF1a shControl knockdown cells demonstrate a colocalization (yellow) of APOBEC3C and ADAR1 p150 proteins in the shControl cells. TF1a shADAR1 cells show ablation of ADAR1 protein.

Article Snippet: Membranes were incubated with 5% BSA in TBS-T for blocking and probed with primary and secondary antibodies diluted in 5% BSA in TBS-T. APOBEC3C lentiviral Vectors Lentiviral human wild-type APOBEC3C (pCDH-EF1-T2A-copGFP) was cloned by Eton Biosciences.

Techniques: RNA Sequencing Assay, Expressing, Mutagenesis, Transduction, Transformation Assay, Western Blot, Immunoprecipitation, Immunofluorescence

Journal: Cell reports

Article Title: Inflammation Driven Deaminase Deregulation Fuels Human Pre-leukemia Stem Cell Evolution

doi: 10.1016/j.celrep.2020.108670

Figure Lengend Snippet: Key Resource Table

Article Snippet: Membranes were incubated with 5% BSA in TBS-T for blocking and probed with primary and secondary antibodies diluted in 5% BSA in TBS-T. APOBEC3C lentiviral Vectors Lentiviral human wild-type APOBEC3C (pCDH-EF1-T2A-copGFP) was cloned by Eton Biosciences.

Techniques: Quantitative RT-PCR, Staining, Luminescence Assay, Recombinant, Software, Expressing, Transduction