Journal: eLife

Article Title: PEAR, a flexible fluorescent reporter for the identification and enrichment of successfully prime edited cells

doi: 10.7554/eLife.69504

Figure Lengend Snippet:

Article Snippet: Plasmids acquired from the non-profit plasmid distribution service Addgene were the following: pCMV-PE2 (#132775) , pAT9624-BEAR-cloning (#162986), pAT9651-BEAR-GFP (#162989), pAT9752-BEAR-mScarlet (#162991), pAT9658-sgRNA-mCherry (#162987), pAT9679-sgRNA-BFP (#162988) , pX330-Flag-wtSpCas9-H840A (#80453), pX330-Flag-dSpCas9 (#92113), and pX330-Flag-wtSpCas9 (#92353) ( ).

Techniques: Recombinant, Sequencing

Journal: Oncotarget

Article Title: STK3 is a therapeutic target for a subset of acute myeloid leukemias

doi: 10.18632/oncotarget.25238

Figure Lengend Snippet: ( A ) Knock-down levels of STK3 protein 3 days post infection are presented for each cell line. The quantitative values corresponding to each band after normalization to a loading control (GAPDH) are given below the western blot image. shScr, shRNA scrambled negative control. shSTK3, shRNA targeting STK3. ( B ) Indicated AML cell lines transduced with either GFP-tagged STK3 shRNA (shSTK3) or GFP-tagged non-target control shRNA (shScr) vectors. Percentage of GFP positive cells was checked by flow cytometry. GFP percentages normalized to day 2 post infection are presented. Changes in the abundance of GFP positive cells are shown over time. ( C ) STK3 knock-down induces apoptosis in MV4:11 cells. Quantitative analyses of Annexin V-positive cells after treatment with STK3 shRNA (shSTK3) or control shRNA (shSrc) in indicated cell lines are shown. Data are presented as mean ± SD. Significance was assessed by means of Student t test; * P < 0.05 ( D ) Expression of apoptosis related proteins after STK3 knock-down examined by western blot assay in MV4:11 and HL60 cells at day 3 post infection is shown. The quantitative values normalized to GAPDH and the control shRNA are given below the western blot images.

Article Snippet: For expression of Cas9 and STK3 sgRNAs from lentiviral plasmids pLCRISPR.EFS.GFP was used (Addgene plasmid # 57818).

Techniques: Knockdown, Infection, Control, Western Blot, shRNA, Negative Control, Transduction, Flow Cytometry, Expressing

Journal: Oncotarget

Article Title: STK3 is a therapeutic target for a subset of acute myeloid leukemias

doi: 10.18632/oncotarget.25238

Figure Lengend Snippet: ( A ) MV4:11 and HL60 cells were transduced with vectors containing two different gRNAs targeting STK3 (sgSTK3#1 and sgSTK3#2) or an empty vector (sgEmpty, negative control). Genomic PCR prepared from cells with indicated treatments in an T7E1 assay are shown. Arrows indicate the size of wild-type PCR products, arrowheads indicate the expected cleavage products of the T7E1 assays. ( B ) Effects of STK3 knock-out in MV4:11 and HL60 cells. Changes in the percentage of GFP+ cells are presented after normalization. GFP percentage was normalized to day 2 post infection and presented as day 0. Data are presented as mean ± SD.

Article Snippet: For expression of Cas9 and STK3 sgRNAs from lentiviral plasmids pLCRISPR.EFS.GFP was used (Addgene plasmid # 57818).

Techniques: Transduction, Plasmid Preparation, Negative Control, Knock-Out, Infection

Journal: Oncotarget

Article Title: STK3 is a therapeutic target for a subset of acute myeloid leukemias

doi: 10.18632/oncotarget.25238

Figure Lengend Snippet: ( A ) Knock-down of STK3 in primary AML cells. Western blots show protein levels 4 days post infection with indicated shRNAs. The quantitative values corresponding to each band after normalization to loading control (GAPDH) are given below the western blot images. ( B ) Phenotypes of STK3 knock-down in primary AMLs. Cells transduced with either GFP-tagged STK3 shRNA (shSTK3) or GFP-tagged non-target control shRNA (shScr) vectors are shown. GFP percentage was normalized to day 2 post infection and presented as day 0. Changes in the GFP percentages are shown at indicated time points. ( C ) Scheme of the experimental design of LTC-IC. Important steps are indicated by arrows. ( D ) Representative microscopic images of leukemic progenitors of AML#24 transduced with indicated shRNAs in LTC-IC assays. GFP positive and negative cobble stones are highlighted by red circles. ( E ) Quantitative analysis of experiment from panel (D). GFP+ colonies were counted in each well ( n = 3 per each shRNA vector). Data are presented as mean ± SD. Significance was assessed by means of Student t test; ** P < 0.01.

Article Snippet: For expression of Cas9 and STK3 sgRNAs from lentiviral plasmids pLCRISPR.EFS.GFP was used (Addgene plasmid # 57818).

Techniques: Knockdown, Western Blot, Infection, Control, Transduction, shRNA, Plasmid Preparation

Journal: Oncotarget

Article Title: STK3 is a therapeutic target for a subset of acute myeloid leukemias

doi: 10.18632/oncotarget.25238

Figure Lengend Snippet: ( A ) In vitro compound screen identified UCN-01 as a strong STK3 binding partner. Each line represents a compound with a temperature shift >3° C. Staurosporine-derivatives with their temperature shift differences are highlighted by their names. The structural formula of UCN-01 is shown on the right. ( B ) Treatment of MV4:11 cells with UCN-01 affects MOB1 phosphorylation. Increasing concentrations of UCN-01 (150 nM and 500 nM) were tested for 4 h and MOB1 phosphorylation was assessed by a western blot assay. Quantification of the p-MOB1 band intensities, normalized to GAPDH, are shown. ( C ) Treatment of primary AML cells with UCN-01 affects MOB1 phosphorylation. Increasing concentrations of UCN-01 (150 nM and 500 nM) were tested for 18 h and MOB1 phosphorylation was assessed by a western blot assay. Quantification of the p-MOB1 band intensities, normalized to GAPDH, are shown.

Article Snippet: For expression of Cas9 and STK3 sgRNAs from lentiviral plasmids pLCRISPR.EFS.GFP was used (Addgene plasmid # 57818).

Techniques: In Vitro, Binding Assay, Phospho-proteomics, Western Blot

Journal: Oncotarget

Article Title: STK3 is a therapeutic target for a subset of acute myeloid leukemias

doi: 10.18632/oncotarget.25238

Figure Lengend Snippet: ( A ) Dose-escalation effect of UCN-01 on selected AML cell lines. Cell survival after treatment with increasing concentrations of UCN-01 for 96 h in indicated AML cell lines is shown. ( B ) Dose-escalation effect of UCN-01 on selected primary AML cells. Cell survival after treatment with increasing concentrations of UCN-01 for 96 h in primary AML cells is shown. The results are presented as percentages of the control condition (only DMSO as a vehicle) for each sample. Data are presented as mean ± SD in A and B panel. ( C ) Expression of apoptosis related proteins after increasing concentration of UCN-01 (150 nM and 500 nM) examined by western blot assay in AML cell lines (MV4:11 and HL60) after 4 h and primary AML cells (AML#29 and AML#31) after 18 h are shown. The quantitative values normalized to GAPDH and the control are given below the western blot images. ( D ) Expression of exogenous STK3 increases phospho-MOB1 (p-MOB1) levels in MV4:11 cells. Increasing multiplicity of infection (MOI) resulted in increased phosphorylation of MOB1. Quantifications of bands are shown below the blot image as a percentage of mock control. ( E ) Over-expression of STK3 affects UCN-01 induced cell death compared to cells transduced with mock vector. Data are presented as mean ± SD. Significance was assessed by means of Student t test; * P < 0.05, ** P < 0.01.

Article Snippet: For expression of Cas9 and STK3 sgRNAs from lentiviral plasmids pLCRISPR.EFS.GFP was used (Addgene plasmid # 57818).

Techniques: Control, Expressing, Concentration Assay, Western Blot, Infection, Phospho-proteomics, Over Expression, Transduction, Plasmid Preparation

Journal: Oncotarget

Article Title: STK3 is a therapeutic target for a subset of acute myeloid leukemias

doi: 10.18632/oncotarget.25238

Figure Lengend Snippet: ( A ) Representative western blot analysis of indicated mitosis related proteins and their respective phosphorylated versions (p-) after STK3 knock-down in AML cell lines (MV4:11 and HL60, respectively). ( B ) Representative western blot analysis of indicated mitosis related proteins and their respective phosphorylated versions (p-) after 4 h treatment with UCN-01 in indicated AML cell lines. ( C ) Representative western blot analysis of indicated mitosis related proteins and their respective phosphorylated versions (p-) after 18 h treatment with UCN-01 in indicated primary AML cells (AML#24 and AML#31, respectively). Quantifications of bands are shown below the blot image as a percentage of scramble control or vehicle (DMSO) treated control. * indicates unspecific bands.

Article Snippet: For expression of Cas9 and STK3 sgRNAs from lentiviral plasmids pLCRISPR.EFS.GFP was used (Addgene plasmid # 57818).

Techniques: Western Blot, Knockdown, Control

Journal: Oncotarget

Article Title: STK3 is a therapeutic target for a subset of acute myeloid leukemias

doi: 10.18632/oncotarget.25238

Figure Lengend Snippet: Sensitive AML cells react to STK3 inhibition/depletion by diminished inhibitory phosphorylation (Y15) of CDK1 and with accumulation of cyclin B1. This aberrant activation of cell cycle proteins leads to apoptosis. Resistant AML cells do not show this effect and survive STK3 inhibition/depletion.

Article Snippet: For expression of Cas9 and STK3 sgRNAs from lentiviral plasmids pLCRISPR.EFS.GFP was used (Addgene plasmid # 57818).

Techniques: Inhibition, Phospho-proteomics, Activation Assay