Journal: bioRxiv

Article Title: TET2 lesions enhance the aggressiveness of CEBPA- mutant AML by rebalancing GATA2 expression

doi: 10.1101/2023.03.28.534511

Figure Lengend Snippet: ( A ) Schematic representation of generation of Tet2 -knockout clones with CRISPR/Cas9. ( B ) Proliferative outgrowth of Cebpa p30/p30 cells with Tet2 indels. ( C ) Volcano plot depicting differentially expressed genes dependent on the Tet2 mutational status in Cebpa p30/p30 cells (n=5–7 per group). ( D ) Experimental setup for evaluating the effect of Tet2 -deficiency ( Tet2 −/− ) in Cebpa DM AML initiation in vivo . ( E ) Myeloid (Mac1 + ) contribution of donor-derived blood and bone marrow (BM) cells evaluated at 12, 24, and 36 weeks after BM transplantation and Cre-LoxP recombination to generate a Cebpa −/p30 and Tet2 −/− hematopoietic compartment (n=3–6 per genotype and timepoint). ( F ) Survival of lethally irradiated recipient mice after BM transplantation and Cre-LoxP recombination (n=12–14/group). ( G ) Volcano plot depicting differentially expressed genes dependent on Tet2 deficiency status in Cebpa −/p30 leukemic blasts (n=3 per group). ( H ) Frequency of proliferating (Ki67 + ) cells in BM of moribund recipient mice (n=3 per group). **=P<0.01, ***=P<0.001, ****=P<0.0001

Article Snippet: For generation of Tet2 or Gata2 mutated clones, Cebpa p30/p30 cells were electroporated with ribonucleoparticles containing recombinant Cas9 nuclease from Streptococcus pyogenes (Sp) (#1081058, IDT), tracrRNA (#1075927, IDT) and crRNAs (Alt-R® CRISPR-Cas9 crRNA, IDT) targeting Tet2 and Gata2 , respectively. crRNAs were designed using the CHOPCHOP web tool (chopchop.cbu.uib.no) ( Supplemental table 4 ). crRNA and tracrRNA molecules were complexed at room temperature and assembled with recombinant SpCas9 according to manufacturer’s protocols (IDT).

Techniques: Knock-Out, Clone Assay, CRISPR, In Vivo, Derivative Assay, Transplantation Assay, Irradiation

Journal: bioRxiv

Article Title: TET2 lesions enhance the aggressiveness of CEBPA- mutant AML by rebalancing GATA2 expression

doi: 10.1101/2023.03.28.534511

Figure Lengend Snippet: ( A ) Experimental setup for evaluating the effect of Gata2 knockdown, via short hairpin RNA (shRNA) mediated silencing, on Cebpa p30/p30 leukemic cells in a competitive in vivo assay. ( B ) Gata2 mRNA in Cebpa p30/p30 leukemic cells prior to transplantation. ( C ) Representative flow cytometry profiles of input and output of shControl (no knockdown), sh Gata2 A (low knockdown), and sh Gata2 D (high knockdown). ( D ) Competitive advantage of targeting shRNA (GFP + ) vs. non-targeting shRNA (YFP + ) cells in vivo assessed as by flow cytometry (n=3–4 per group). ( E ) Experimental setup for Gata2 CRISPR/Cas9 mutagenesis in Cebpa p30/p30 cells, and outgrowth of heterozygous mutated clones. Percentages of Gata2 mutated clones are indicated. ( F ) Growth curve of Cebpa p30/p30 clones with Gata2 mutation ( Cebpa p30/p30 Gata2 +/MUT ) or wild type Gata2 ( Cebpa p30/p30 Gata2 +/+ ). Red lines mark individual clones. (G) Presence or absence of GATA2 mutations ( GATA2 MUT ) in CEBPA double mutated ( CEBPA DM ) AML cases (n=460) with or without TET2 mutations ( TET2 MUT ) in aggregated data from published cohorts – , , , . *=P<0.05, **=P<0.01

Article Snippet: For generation of Tet2 or Gata2 mutated clones, Cebpa p30/p30 cells were electroporated with ribonucleoparticles containing recombinant Cas9 nuclease from Streptococcus pyogenes (Sp) (#1081058, IDT), tracrRNA (#1075927, IDT) and crRNAs (Alt-R® CRISPR-Cas9 crRNA, IDT) targeting Tet2 and Gata2 , respectively. crRNAs were designed using the CHOPCHOP web tool (chopchop.cbu.uib.no) ( Supplemental table 4 ). crRNA and tracrRNA molecules were complexed at room temperature and assembled with recombinant SpCas9 according to manufacturer’s protocols (IDT).

Techniques: Knockdown, shRNA, In Vivo, Transplantation Assay, Flow Cytometry, CRISPR, Mutagenesis, Clone Assay

Journal: bioRxiv

Article Title: TET2 lesions enhance the aggressiveness of CEBPA- mutant AML by rebalancing GATA2 expression

doi: 10.1101/2023.03.28.534511

Figure Lengend Snippet: ( A ) Gata2 mRNA expression in mouse Cebpa p30/p30 leukemic granulocyte/monocyte progenitors (GMPs) vs normal GMPs and, ( B ) CEBPA binding to the Gata2 distal hematopoietic enhancer ( G2 DHE; −77kb) region, data from Jakobsen et al. (n=2–4 per group). ( C ) Schematic genomic view of the Gata2 distal hemeatopoietic enhancer ( G2 DHE), including tracks from CEBPA and H3K27Ac chromatin immunoprecipitation sequencing (ChIP-seq) in Cebpa p /p30 cells (data from Heyes et al. ), TET2 ChIP-seq in AML-ETO expressing cells (data from Rasmussen et al. ),Targeting of the G2 DHE by dual-( D ) or single-( E ) guided CRISPR-Cas9 in Cebpa p30/p30 cells using indicated sgRNAs (n=3/condition). ( F ) Experimental setup for evaluating the effects of Cebpa knockout on Gata2 V2 mRNA expression and DNA methylation of the CpG island at the promoter of Gata2 V2 in MLL-fusion driven AML ( iMLL-AF9 ). ( G ) Cebpa and ( H ) Gata2 V2 mRNA expression upon induction of Cre-LoxP recombination and, ( I ) DNA methylation of the Gata2 V2 promoter CpG-island (2 biological replicates per genotype). ( J ) Frequency of GATA2 and/or TET2 mutations ( GATA2 MUT and TET2 MUT , respectively) in CEBPA high expressing ( CEBPA HIGH n=45) vs. CEBPA low expressing (CEBPA LOW n=61) AML cases, data from Beat AML cohort . *=P<0.05, **=P<0.01, ***=P<0.001, ****=P<0.0001

Article Snippet: For generation of Tet2 or Gata2 mutated clones, Cebpa p30/p30 cells were electroporated with ribonucleoparticles containing recombinant Cas9 nuclease from Streptococcus pyogenes (Sp) (#1081058, IDT), tracrRNA (#1075927, IDT) and crRNAs (Alt-R® CRISPR-Cas9 crRNA, IDT) targeting Tet2 and Gata2 , respectively. crRNAs were designed using the CHOPCHOP web tool (chopchop.cbu.uib.no) ( Supplemental table 4 ). crRNA and tracrRNA molecules were complexed at room temperature and assembled with recombinant SpCas9 according to manufacturer’s protocols (IDT).

Techniques: Expressing, Binding Assay, ChIP-sequencing, CRISPR, Knock-Out, DNA Methylation Assay

Journal: Aging and Disease

Article Title: Mash1-dependent Notch Signaling Pathway Regulates GABAergic Neuron-Like Differentiation from Bone Marrow-Derived Mesenchymal Stem Cells

doi: 10.14336/AD.2016.1018

Figure Lengend Snippet: Primer sequences for Notch signaling and GABAergic neuron marker

Article Snippet: For RBPJ or Hes1 silencing, BMSCs which reached approximately 50% confluence in a 12-well plate were replaced with Polybrene (Santa Cruz Biotechnology, Inc., sc-134220, CA, USA) media mixture, followed by infection with RBPJ (Santa Cruz Biotechnology, Inc., sc-270318-v, CA, USA) or Hes1 (Santa Cruz Biotechnology, Inc., sc-270146-v, CA, USA) shRNA lentiviral particles (these primers were provided by the company) overnight (37°C, 5% CO 2 ).

Techniques: Sequencing

Journal: Aging and Disease

Article Title: Mash1-dependent Notch Signaling Pathway Regulates GABAergic Neuron-Like Differentiation from Bone Marrow-Derived Mesenchymal Stem Cells

doi: 10.14336/AD.2016.1018

Figure Lengend Snippet: A ) Protein bands of Notch signaling (RBPJ, Hes1 and Mash1) visualized through western blotting of BMSCs that were treated by DAPT (DAPT+BMSCs), or engineered by Hes1 shRNA (Hes1-BMSCs), pGC-FU-Mash1 plasmid (Mash1+BMSCs), RBPJ shRNA (RBPJ-BMSCs) and copGFP Control (Lv-con-BMSCs). B ) The protein level based on the ratio of Gauss Model Trace exhibited the Notch signaling (RBPJ, Hes1 and Mash1) changes in the DAPT+ BMSCs. C ) The protein level based on the ratio of Gauss Model Trace exhibited genetically engineered BMSCs. Error bars in bar graphs display standard deviation (SD). * P = 0.032, < 0.05, ** P = 0.0006, < 0.01.

Article Snippet: For RBPJ or Hes1 silencing, BMSCs which reached approximately 50% confluence in a 12-well plate were replaced with Polybrene (Santa Cruz Biotechnology, Inc., sc-134220, CA, USA) media mixture, followed by infection with RBPJ (Santa Cruz Biotechnology, Inc., sc-270318-v, CA, USA) or Hes1 (Santa Cruz Biotechnology, Inc., sc-270146-v, CA, USA) shRNA lentiviral particles (these primers were provided by the company) overnight (37°C, 5% CO 2 ).

Techniques: Western Blot, shRNA, Plasmid Preparation, Control, Standard Deviation

Journal: PLoS ONE

Article Title: Differential Proteomic Analysis of Human Erythroblasts Undergoing Apoptosis Induced by Epo-Withdrawal

doi: 10.1371/journal.pone.0038356

Figure Lengend Snippet: Proteins with altered abundance in SDL with a change in average ratio of >2 and a t -test of p <0.05.

Article Snippet: Primary antibodies used (with catalog numbers in brackets) were Caspase 8 (1C12, 9746), Caspase 9 (9502), cleaved Caspase 3 (9664), Hsp90beta (5087) and Lamin A/C (2032) from Cell Signalling Technology; Hsp90alpha (mAb 9D2, SPA-840) from Enzo/Stressgen; Actin (sc-1616 rabbit), RPSA (Laminin-R (16), sc-101517) and SET (I2PP2A, sc-5655) from Santa Cruz; Cytochrome C (Clone 7H8.2C12, 556443) from BD Pharmingen; Bax (anti-Bax NT, 06–499), 14-3-3 beta (AB9730), 14-3-3 epsilon (clone CG31-2B6, 05-639) and 14-3-3 gamma (AB9734) from Millipore/Upstate Cell Signalling and Hsc70 (ab19136) from Abcam.

Techniques:

Journal: PLoS ONE

Article Title: Differential Proteomic Analysis of Human Erythroblasts Undergoing Apoptosis Induced by Epo-Withdrawal

doi: 10.1371/journal.pone.0038356

Figure Lengend Snippet: Western blotting of total cell lysates harvested from one independent culture after 6 hour, 12 hour and 24 hour in ESDL (+Epo) and SDL (-Epo) using antibodies against SET, 14-3-3 β, 14-3-3 γ, 14-3-3 ε, RPSA, Hsp90 isofoms alpha and beta. 20 µg of protein lysate was loaded per lane. Beta Actin and Hsc70 were used as loading controls. The arrows point to the smaller proteolytic fragments that occur in apoptotic erythroblasts.

Article Snippet: Primary antibodies used (with catalog numbers in brackets) were Caspase 8 (1C12, 9746), Caspase 9 (9502), cleaved Caspase 3 (9664), Hsp90beta (5087) and Lamin A/C (2032) from Cell Signalling Technology; Hsp90alpha (mAb 9D2, SPA-840) from Enzo/Stressgen; Actin (sc-1616 rabbit), RPSA (Laminin-R (16), sc-101517) and SET (I2PP2A, sc-5655) from Santa Cruz; Cytochrome C (Clone 7H8.2C12, 556443) from BD Pharmingen; Bax (anti-Bax NT, 06–499), 14-3-3 beta (AB9730), 14-3-3 epsilon (clone CG31-2B6, 05-639) and 14-3-3 gamma (AB9734) from Millipore/Upstate Cell Signalling and Hsc70 (ab19136) from Abcam.

Techniques: Western Blot