Journal: Oncotarget

Article Title: Decreased calpain activity in chronic myeloid leukemia impairs apoptosis by increasing survivin in myeloid progenitors and xiap1 in differentiating granulocytes

doi: 10.18632/oncotarget.16884

Figure Lengend Snippet: ( A ) Either Bcr-abl expression or Icsbp knockout increased Gas2 mRNA in myeloid progenitor cells and Calpastatin mRNA in differentiating granulocytes. Bone marrow cells from wild type and Icsbp −/− mice were compared; some wild type cells were transduced with a Bcr-abl-expression vector. Lin − CD34 + cells were analyzed for Gas2 or Calpastatin mRNA by real time PCR with or without G-CSF-differentiation. Statistically significant differences ( p < 0.01) in mRNA are indicated by *, **, ***, #, ## or ###. Non-significant differences are indicated by p value on the figure. Lysates from these cells were analyzed for protein expression by serially probing Western blots with antibodies to Calpastatin, Gas2 or Gapdh (loading control). Icsbp knockout ( B ) or Bcr-abl expression ( C ) increased Gas2 protein in myeloid progenitor cells and Calpastatin protein in differentiating granulocytes relative to control. ( D ) Gas2 expression was increased in human Lin − CD34 + CML cells, but Calpastatin was increased in differentiating CML granulocytes, in comparison to control. Lin − CD34 + bone marrow cells from CML or control subjects were analyzed for mRNA expression by real time PCR with or without G-CSF-differentiation. Statistically significant differences ( p < 0.01) in mRNA were indicated by *, **, ***, #, ## or ###. Non-significant differences were indicated by ‘a’ or ‘b’.

Article Snippet: Lin − CD34 + normal human bone marrow cells purchased from Stem Cell Technologies (Vancouver, Canada) to use as controls for these studies.

Techniques: Expressing, Knock-Out, Transduction, Plasmid Preparation, Real-time Polymerase Chain Reaction, Western Blot, Control, Comparison

Journal: Oncotarget

Article Title: Decreased calpain activity in chronic myeloid leukemia impairs apoptosis by increasing survivin in myeloid progenitors and xiap1 in differentiating granulocytes

doi: 10.18632/oncotarget.16884

Figure Lengend Snippet: Cells populations used in the experiments above were analyzed by immuno-phenotyping. Representative histograms are presented for ( A ). murine Bcr-abl transduced Lin − CD34 + bone marrow cells, or ( B ) human Lin − CD34 + bone marrow mononuclear cells from a CML subject in chronic phase.

Article Snippet: Lin − CD34 + normal human bone marrow cells purchased from Stem Cell Technologies (Vancouver, Canada) to use as controls for these studies.

Techniques:

Journal: Oncotarget

Article Title: Decreased calpain activity in chronic myeloid leukemia impairs apoptosis by increasing survivin in myeloid progenitors and xiap1 in differentiating granulocytes

doi: 10.18632/oncotarget.16884

Figure Lengend Snippet: ( A ) Icsbp repressed a cis element between −0.5 and −1.0 kb in the CAST 5′ flank. U937 cells were co-transfected with reporter constructs with various lengths of CAST 5′ flank and a vector to express Icsbp or control. Transfectants were assayed for reporter activity with or without granulocyte differentiation (with RA + DMF). Statistically significant differences ( p < 0.01) were indicated by *, **, ***, #, or ##. ( B ) CAST promoter activity was repressed by Bcr-abl in an Icsbp-dependent manner and Icsbp repression required IRF-domain tyrosine residues. U937 cells were co-transfected with the −1.0 kb CAST promoter/reporter construct and vectors to express various combinations of Icsbp (wild type or Y-mutant) and Bcr-abl (or relevant control vectors). Reporter activity was determined as above. Statistically significant differences in reporter activity ( p < 0.01) were indicated by *, **, ***, # or ##. Non-significant differences were indicated by ‘a’. ( C ) Bcr-abl decreased Icsbp binding to the CAST promoter. U937 cells were transfected with a vector to express Bcr-abl or Icsbp (or control vector) and analyzed by chromatin immunoprecipitation (ChIP) using antibody to Icsbp or irrelevant control antibody. Co-precipitating chromatin was amplified by real time PCR with primers flanking the CAST promoter sequence between −0.5 and −1.0 kb. Statistically significant differences ( p < 0.01) were indicated by * or **. ( D ) Icsbp binding to the CAST promoter increased during granulocyte differentiation. CD34 + murine bone marrow cells were analyzed by ChIP with or without G-CSF-differentiation. Lysates were immuno-precipitated with an antibody to Icsbp or irrelevant control antibody, and co-precipitating chromatin was amplified by real time PCR using primers flanking Icsbp-binding cis elements in the CAST or GAS2 genes. Statistically significant differences ( p < 0.01) were indicated by * or**.

Article Snippet: Lin − CD34 + normal human bone marrow cells purchased from Stem Cell Technologies (Vancouver, Canada) to use as controls for these studies.

Techniques: Transfection, Construct, Plasmid Preparation, Control, Activity Assay, Mutagenesis, Binding Assay, Chromatin Immunoprecipitation, Amplification, Real-time Polymerase Chain Reaction, Sequencing

Journal: Oncotarget

Article Title: Decreased calpain activity in chronic myeloid leukemia impairs apoptosis by increasing survivin in myeloid progenitors and xiap1 in differentiating granulocytes

doi: 10.18632/oncotarget.16884

Figure Lengend Snippet: ( A ) Calpastatin knockdown rescued Calpain activity in Bcr-abl + or Icsbp −/− myeloid progenitors undergoing granulocyte differentiation. Wild type or Icsbp −/− murine bone marrow cells were transduced with vectors to express Calpastatin-specific shRNAs (or scrambled shRNA control). Some wild type cells were also transduced with a Bcr-abl expression vector. Lin - CD34 + cells were analyzed for Calpain activity with or without G-CSF-differentiation. Statistically significant differences ( p < 0.02) were indicated by *, **, ***, #, ##, ###, & or &&, and non-significant differences by ‘a’. ( B ) Gas2 knockdown normalized Calpain activity in Lin − CD34 + CML cells and Calpastatin knockdown normalized Calpain activity in differentiating CML granulocytes. Human Lin − CD34 + bone marrow cells from CML or normal subjects were transduced with a retroviral vector to express Gas2 or Calpastatin specific shRNAs (or scrambled control). Cells were analyzed for Calpain activity with or without ex vivo G-CSF differentiation. Statistically significant differences ( p < 0.01) were indicated by *, ** or ***, and non-significant differences by ‘a’ or ‘b’. ( C ) Knockdown of Gas2 increased βcatenin and Survivin protein in Bcr-abl-transduced myeloid progenitors. Murine bone marrow cells were transduced with vectors to express Bcr-abl (or control) and Gas2 or Calpastatin specific shRNAs. Lin − CD34 + cell lysates were analyzed by Western blots serially probed with antibodies to βcatenin, Survivin, Xiap1, Gas2, Calpastatin or Gapdh (loading control). ( D ) Some cells were similarly analyzed after G-CSF-differentiation. ( E ) Knockdown of Gas2 increased Survivin mRNA in Bcr-abl transduced murine myeloid progenitor cells. The cells described in ‘C’ and ‘D’ were also analyzed for mRNA expression by real time PCR. Statistically significant differences ( p < 0.02) are indicated by *, **, *** or #.

Article Snippet: Lin − CD34 + normal human bone marrow cells purchased from Stem Cell Technologies (Vancouver, Canada) to use as controls for these studies.

Techniques: Knockdown, Activity Assay, Transduction, shRNA, Control, Expressing, Plasmid Preparation, Retroviral, Ex Vivo, Western Blot, Real-time Polymerase Chain Reaction

Journal: Oncotarget

Article Title: Decreased calpain activity in chronic myeloid leukemia impairs apoptosis by increasing survivin in myeloid progenitors and xiap1 in differentiating granulocytes

doi: 10.18632/oncotarget.16884

Figure Lengend Snippet: Wild type or Icsbp −/− murine bone marrow cells were transduced with a vector to express a shRNA specific to Calpastatin, Xiap1 or Survivin (or scrambled shRNA control). Some wild type cells were co-transduced with a Bcr-abl-expression vector (or control). Lin − CD34 + cells were analyzed for apoptosis by Annexin V staining, with or without G-CSF-differentiation, with or without Fas-agonist antibody. ( A ) Knockdown of Calpastatin increased sensitivity to intrinsic and Fas-induced apoptosis in differentiating Bcr-abl transduced or Icsbp −/− myeloid progenitor cells. Statistically significant differences ( p < 0.02) were indicated by *, **, *** or #. ( B ) Knockdown of Xiap1 increased sensitivity to intrinsic and Fas-induced apoptosis in differentiating Bcr-abl transduced or Icsbp −/− myeloid progenitors. Statistically significant differences ( p < 0.02) were indicated by *, **, *** or #. ( C ) Knockdown of Survivin increased sensitivity to intrinsic and Fas-induced apoptosis in Bcr-abl transduced or Icsbp -/- myeloid progenitors. Statistically significant differences ( p < 0.02) were indicated by *, **, *** or #. ( D ) Imatinib (IM) enhanced the effect of Survivin knockdown on apoptosis in Bcr-abl-transduced murine myeloid progenitor cells. Some Lin − CD34 + cells transduced with vectors to express Bcr-abl and shRNA to Xiap1 or Survivin (or scrambled control vector) were treated with IM prior to analysis. Statistically significant differences ( p < 0.02) were indicated by *, **, ***, # or ##. ( E ) IM enhanced effects of Gas2 knockdown on apoptosis in human Lin − CD34 + CML bone marrow cells. Human Lin − CD34 + bone marrow cells from CML or control subjects were transduced with vectors to express shRNA specific to calpastatin or Gas2 (or scrambled shRNA control) and analyzed for apoptosis, with or without treatment with IM, and with or without Fas-agonist. Statistically significant differences ( p < 0.02) were indicated by *, **, ***, #, ##, ###, &, &&, &&&, ^, ^^, ^^^, @ or @@.

Article Snippet: Lin − CD34 + normal human bone marrow cells purchased from Stem Cell Technologies (Vancouver, Canada) to use as controls for these studies.

Techniques: Transduction, Plasmid Preparation, shRNA, Control, Expressing, Staining, Knockdown