particles expressing cas9  (Beyotime)

Journal: Antioxidants

Article Title: Combined Activity of the Redox-Modulating Compound Setanaxib (GKT137831) with Cytotoxic Agents in the Killing of Acute Myeloid Leukemia Cells

doi: 10.3390/antiox11030513

Figure Lengend Snippet: Assessment of a potential mechanism for Setanaxib effects. ( A , B ) Effect of Setanaxib on the proliferation of human AML cells with NOX4 or p22-phox knockout. KO human AML cells were obtained using CRISPR/Cas9 technology. The cell lines stably express Cas9 and were either transduced with control sgRNA (sgLuci, designated WT) or sgRNA targeting NOX4 or p22-phox , respectively (designated KO). Absence of the targeted gene was detected as described in Materials and Methods. The experimental setup for the drug treatments and assay of proliferation were as in A,B. Cells were counted using a hemocytometer at day 8. Mean ± SD of cell treatments of two to three independently sgRNA-transduced cell batches is presented. ( C ) Comparison of synergy of Setanaxib with daunorubicin in Ba/F3-FLT3-ITD cells harboring Cas9 and transduced with sgLuci (WT) or sg Nox4 (KO). Treatments were performed as in and proliferation/viability was assessed by Cell Titer Blue assay. The individual experiments were normalized to DMSO controls. Mean ± SD (with technical triplicates) for cells from three independent transductions with sgRNA is shown. ( D , E ) The 32D-FLT3-ITD cells were subjected to single or combined drug treatments with daunorubicin, diphenyleneiodonium (DPI) or N-acetylcysteine (NAC) as indicated for 72 h, and proliferation/viability was assessed by Cell Titer Blue assay. Three independent experiments (in triplicate) were conducted; error bars represent mean ± SD. Statistical analyses were carried out using two-tailed t -test (n.s.—not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001).

Article Snippet: In brief, MOLM13, MV4-11, Ba/F3 cells, and HEK293 cells (with tet-inducible NOX4) with stable Cas9 expression were generated by transduction with lentiviral particles expressing Cas9 (Streptococcus pyogenes gene in lentiCas9-Blast plasmid, #52962, Addgene Cambridge, MA, USA) and cell selection with blasticidin using standard techniques, and subsequent clonal selection was employed for high Cas9 levels, monitored by immunoblotting.

Techniques: Knock-Out, CRISPR, Stable Transfection, Transduction, Control, Comparison, Two Tailed Test

Journal: Antioxidants

Article Title: Combined Activity of the Redox-Modulating Compound Setanaxib (GKT137831) with Cytotoxic Agents in the Killing of Acute Myeloid Leukemia Cells

doi: 10.3390/antiox11030513

Figure Lengend Snippet: Setanaxib promotes ROS formation elicited by daunorubicin. ( A – C ) MV4-11 cells were treated with the indicated concentrations of the FLT3-ITD inhibitor AC220 (quizartinib, positive control), the general NOX inhibitor diphenyleneiodonium (DPI), or Setanaxib for 4 h in serum-free medium. Thereafter, cells were lysed and lysates subjected to immunoblotting for assessment of pathway activation. Blots were probed with activation-specific antibodies to pFLT3 (pY589/591), pSTAT5 (pY694), pAkt (Ser473), or pErk1/2 (Thr202/Tyr204) antibodies. Subsequently, blots were stripped and comparable loading was validated by reprobing of the membranes with antibodies against FLT3, STAT5, Akt, or Erk as indicated. ( A ) Representative result. ( B , C ) Quantification of three independent experiments for the indicated signaling molecules. Setanaxib (GKT137831) is abbreviated as GKT. Error bars represent mean ± SD. Statistical analyses were carried out with two-tailed t -test. ( D ) MOLM-13 cells were treated with 5 mM NAC (positive control), 500 nM of the general NOX inhibitor diphenyleneiodonium (DPI), 40 nM of the protein kinase inhibitor midostaurin, or 10 or 30 µM Setanaxib for 24 h. Thereafter, cells were stained with H 2 DCFDA for 30 min and ROS levels were quantified on the flow cytometer (MFI, mean fluorescence intensity). Error bars represent mean ± SD, ( n = 3). Statistical analyses were carried out using two-tailed t -test. ( E ) The 32D-FLT3-ITD cells were treated with the indicated concentrations of the drugs for 24 h. Thereafter, cells were stained with ROS Deep Red dye for 30 min and ROS levels were quantified by flow cytometry. The experiment was conducted three times independently. Error bars represent mean ± SD, ( n = 3). Statistical analyses were carried out using two-tailed t -test. ( F ) HEK293 cells with tetracycline (tet)-inducible NOX4 overexpression and engineered to constitutively express Cas9 were kept uninduced (−tet) or were induced (+tet) to overexpress NOX4 and were mock-treated with solvent or were treated with Setanaxib as indicated. ROS formation was scored with H 2 DCFDA as in ( D ). Error bars represent mean ± SD, ( n = 3). Statistical analyses were carried out using two-tailed t -test (n.s.—not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001).

Article Snippet: In brief, MOLM13, MV4-11, Ba/F3 cells, and HEK293 cells (with tet-inducible NOX4) with stable Cas9 expression were generated by transduction with lentiviral particles expressing Cas9 (Streptococcus pyogenes gene in lentiCas9-Blast plasmid, #52962, Addgene Cambridge, MA, USA) and cell selection with blasticidin using standard techniques, and subsequent clonal selection was employed for high Cas9 levels, monitored by immunoblotting.

Techniques: Positive Control, Western Blot, Activation Assay, Two Tailed Test, Staining, Flow Cytometry, Fluorescence, Over Expression, Solvent

Journal: Nature cancer

Article Title: Mutational and functional genetics mapping of chemotherapy resistance mechanisms in relapsed acute lymphoblastic leukemia

doi: 10.1038/s43018-020-00124-1

Figure Lengend Snippet: a, Hierarchical cluster analysis of gene-drug interactions from genome-wide CRISPR screens in REH cells treated with different chemotherapy drugs. b, Principal component analysis (PCA) of gene-drug interactions as in a. Each drug feature is plotted based on the top contributing genes to each of the first 2 principle components. Genes with enrichment (positive or negative) greater than 0.5 log fold-change and false discovery rate less than 5% were used in these analyses. c, Circos plot representation of overlapping genes with gRNAs enriched and depleted across different chemotherapy selection-based CRISPR screens. d, Schematic representation of PI3K-mTOR pathway drug-gene interactions identified in genome wide CRISPR screens. Red and blue circles in indicate genes with enriched and depleted gRNAs (FDR < 0.05), respectively. Drug interactions for each gene are in colored arch segments as indicated. e, Western blot analysis of TP53 inactivation and in vitro analyses of the antileukemic responses of REH and RCH control and CRISPR TP53 knockout cells treated with chemotherapy drugs. f, Circos plot representation of genes with divergent gRNA selection profiles across CRISPR screens with different chemotherapeutic drugs. Green links indicate sensitivity; red links indicate resistance. 6-MP: 6-mercaptopurine; AraC: cytarabine; MTX: methotrexate; LASP: L-asparaginase, DNR: daunorubicin; VCR: vincristine; MAF: maphosphamide; (S): drug-sensitizing; (R): drug resistance. Graphs indicate relative cell viability compared to vehicle treated controls.

Article Snippet: We performed gene inactivation of TP53 in REH and RCH cells by lentiviral infection with lentiCRISPR v2 (Addgene 52961) lentiviral particles expressing CAS9 and a gRNA targeting the TP53 locus.

Techniques: Genome Wide, CRISPR, Selection, Western Blot, In Vitro, Control, Knock-Out