Journal: Cancers

Article Title: AMP-Activated Protein Kinase Contributes to Apoptosis Induced by the Bcl-2 Inhibitor Venetoclax in Acute Myeloid Leukemia

doi: 10.3390/cancers13235966

Figure Lengend Snippet: Inhibition of AMPK activity by venetoclax in AML cells. ( A ) Four different AML cell lines were seeded in 384-well plates at 3 × 10 5 cells/mL and incubated with a dose range of venetoclax during 24 h. Cell viability was then measured using the ATPlite luminescent reagent, and results were analyzed with the nonlinear regression module and plotted using log(inhibitor) versus response (three parameters) function of the Prism software. ( B ) Four different AML cell lines were seeded in 384-well plates at 3 × 10 5 cells/mL and incubated with 100 nM venetoclax during 0 h o 8 h. Then, ATP content was measured using the ATP lite luminescent reagent. Results of ATP content (Y-axis) were plotted on incubation time (X-axis) and a linear regression was performed using the Prism software. Time for achieving a 50% reduction of ATP content is indicated by vertical dashed lines for each cell lines following their respective color code. ( C ) AML cell lines were seeded at 5 × 10 5 cells/mL and incubated with 100 nM venetoclax for the indicated times. Western blots were performed using the anti-phospho-AMPKα T172, -AMPKα, -Bcl-2, and -β-actin antibodies. ( D ) AML cell lines were incubated with vehicle (DMSO) or 100 nM venetoclax (VEN) for 6 h, and Western blots were performed using anti-phospho-ACC S79, -phospho-ULK-1 S555, -phospho-AMPKα T172, -AMPKα, and -β-actin antibodies.

Article Snippet: We used the OCI-AML2, HL-60, THP-1, and MOLM-14 AML cell lines, which were identified by PCR single-locus technology (Promega, PowerPlex21 PCR Kit, Eurofins Genomics, Nantes, France).

Techniques: Inhibition, Activity Assay, Incubation, Software, Western Blot

Journal: Cancers

Article Title: AMP-Activated Protein Kinase Contributes to Apoptosis Induced by the Bcl-2 Inhibitor Venetoclax in Acute Myeloid Leukemia

doi: 10.3390/cancers13235966

Figure Lengend Snippet: Decreased amount of AMPK subunits by venetoclax in AML. ( A ) Western blots were done from in vitro kinase assay protein mix using anti-phospho-ACC S79 and anti-phospho-AMPK T172 antibodies. ( B , C ) AML cell lines were incubated without or with 100 nM venetoclax (VEN), and then submitted to a cycloheximide pulse during the indicated times. ( B ) Western blots were done using anti-AMPKα, β, γ, -Bcl-2, and -β-actin antibodies. ( C ) Quantification of the Western blot signals from three independent experiments using ImaJ software for AMPKα detection in the control (CTR) or venetoclax (VEN) conditions.

Article Snippet: We used the OCI-AML2, HL-60, THP-1, and MOLM-14 AML cell lines, which were identified by PCR single-locus technology (Promega, PowerPlex21 PCR Kit, Eurofins Genomics, Nantes, France).

Techniques: Western Blot, In Vitro, Kinase Assay, Incubation, Software, Control

Journal: Cancers

Article Title: AMP-Activated Protein Kinase Contributes to Apoptosis Induced by the Bcl-2 Inhibitor Venetoclax in Acute Myeloid Leukemia

doi: 10.3390/cancers13235966

Figure Lengend Snippet: AMPK degradation is due to on-target caspase activation by venetoclax. ( A ) AML cell lines were incubated with 100 nM venetoclax during the indicated times and processed for flow cytometry using annexin V and DAPI staining. Annexin V-positive and DAPI-negative cells are those in early apoptosis, while double positivity indicates either late apoptosis or necrotic cells. The experiment was repeated three times separately. Vertical bars indicate standard deviations. * p < 0.05, *** p < 0.001. ( B , C ) AML cell lines were incubated with vehicle (CTR), 100 nM venetoclax (VEN), 50 µM Z-VAD (pan-caspase inhibitor) or a combination of 50 µM Z-VAD (added 24 h before VEN) and 100 nM venetoclax for 4 h. ( B ) Flow cytometry measurement of annexin V binding done in three separate experiments and plotted in a heat-map format. ( C ) Western blots done using anti-phospho-AMPK T172, -AMPK α, β, γ, -cleaved caspase 3, and –β-actin.

Article Snippet: We used the OCI-AML2, HL-60, THP-1, and MOLM-14 AML cell lines, which were identified by PCR single-locus technology (Promega, PowerPlex21 PCR Kit, Eurofins Genomics, Nantes, France).

Techniques: Activation Assay, Incubation, Flow Cytometry, Staining, Binding Assay, Western Blot

Journal: Cell stem cell

Article Title: The Mitochondrial Transacylase, Tafazzin, Regulates AML Stemness by Modulating Intracellular Levels of Phospholipids

doi: 10.1016/j.stem.2019.02.020

Figure Lengend Snippet: (A) Results of a dropout screen in Cas9-OCI-AML2 cells. Positive-hits were identified at a false discovery rate (FDR) <5%. (B) The rank of TAZ in screens of OCI-AML2, OCI-AML3, MOLM-13, MV4–11, and HL-60 cells from the published CRISPR dropout screens by Tzelepis et al. (2016). (C) A model of the enzymatic steps involved in cardiolipin synthesis and remodeling, where TAZ utilizes phosphatidylcholine (PC) or phosphatidylethanolamine (PE) as acyl chain donors to reacylate monolysocardiolipin (MLCL). (D) Proliferation of CAS-9-OCI-AML2 cells after CRISPR-mediated knockout of TAZ. The relative area under the curve (AUC) of viable cell counts over 12 days are shown. Control sgRNA = 100%. Data are mean ± SEM (N = 3). ****p ≤ 0.0001 by one-way ANOVA and Dunnett’s post hoc test. (E and F) Proliferation and TAZ protein expression of OCI-AML2 (E) and TEX cells (F) after shRNA-mediated TAZ knockdown. The relative AUC of viable cell counts over 12 days are shown (control shRNA = 100%). Data are mean ± SD (n = 2) of a representative experiment from 3 independent experiments. **p ≤ 0.01, ***p ≤ 0.001 by one-way ANOVA and Dunnett’s post hoc test. See also Figure S1 and Table S1.

Article Snippet: . . Leukemia Cell Lines Five (TAZ knockdown OCI-AML2, and TEX cells) or nine (PISD knockout CAS9-OCI-AML2 cells) days after transduction cells were plated at equal concentrations (CAS9-OCI-AML2 and OCI-AML2 = 750 cells; TEX cells = 2,000) in duplicate 35mm dishes. (Nunclon, Rochester, USA) to a final volume of 1 mL/dish in MethoCult H4100 media (StemCell Technologies, BC, Canada) supplemented with 30% FCS (CAS9-OCI-AML2 and OCI-AML2) or MethoCult H4100 media (StemCell Technologies, BC, Canada) supplemented with 30% FCS, 20 ng/mLSCF, and 2 ng/mL IL-3 (TEX cells).

Techniques: CRISPR, Knock-Out, Control, Expressing, shRNA, Knockdown

Journal: Cell stem cell

Article Title: The Mitochondrial Transacylase, Tafazzin, Regulates AML Stemness by Modulating Intracellular Levels of Phospholipids

doi: 10.1016/j.stem.2019.02.020

Figure Lengend Snippet: (A) Heatmap of standardized Z score expression of the 500 most highly upregulated genes in OCI-AML2 cells after TAZ shRNA knockdown. Rows represent genes and columns represent LSC+ (pink bars) or LSC− (black bars) fractions. (B) Gene set enrichment analysis (GSEA) of OCI-AML2 cells from (A). The normalized enrichment scores (NES), and false discovery rates (FDRs) are indicated in each GSEA plot. (C) Non-specific esterase (NSE) staining of OCI-AML2 cells after TAZ shRNA knockdown. Data are relative mean ± SEM (N = 4, control shRNA = 1.0 ABU). ****p ≤ 0.0001 by one-way ANOVA and Dunnett’s post hoc test. (D and E) Clonogenic growth of OCI-AML2 (D) or TEX (E) after TAZ shRNA knockdown. Data are relative mean ± SEM (N = 3, control shRNA = 100%). ****p ≤ 0.0001 by one-way ANOVA and Dunnett’s post hoc test. (F) CD45+TEXcell engraftment after TAZ shRNA knockdown. Bar represents mean (n = 10 mice control shRNA group, n = 9 mice TAZ shRNA2 group). **p ≤ 0.01 by Student’s t test. See also Figure S2.

Article Snippet: . . Leukemia Cell Lines Five (TAZ knockdown OCI-AML2, and TEX cells) or nine (PISD knockout CAS9-OCI-AML2 cells) days after transduction cells were plated at equal concentrations (CAS9-OCI-AML2 and OCI-AML2 = 750 cells; TEX cells = 2,000) in duplicate 35mm dishes. (Nunclon, Rochester, USA) to a final volume of 1 mL/dish in MethoCult H4100 media (StemCell Technologies, BC, Canada) supplemented with 30% FCS (CAS9-OCI-AML2 and OCI-AML2) or MethoCult H4100 media (StemCell Technologies, BC, Canada) supplemented with 30% FCS, 20 ng/mLSCF, and 2 ng/mL IL-3 (TEX cells).

Techniques: Expressing, shRNA, Knockdown, Staining, Control

Journal: Cell stem cell

Article Title: The Mitochondrial Transacylase, Tafazzin, Regulates AML Stemness by Modulating Intracellular Levels of Phospholipids

doi: 10.1016/j.stem.2019.02.020

Figure Lengend Snippet: (A and B) The relative MLCL:CL ratio (A) and cardiolipin double bonds (B) in OCI-AML2 cells after TAZ knockdown. Data are mean ± SEM (N = 3). *p ≤ 0.05, ***p ≤ 0.001, ****p ≤ 0.0001 by Student’s t test (ML:CL ratio) or two-way ANOVA and post hoc Dunnett’s test (cardiolipin double bonds). (C) Composition of sphingomyelin (SM), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylethanolamine (PE), and cardiolipin (CL) in OCI-AML2 cells after TAZ knockdown. Data are mean ± SEM (N = 3). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 by two-way ANOVA, and Dunnett’s post hoc test. (D) Extracellular PS levels in OCI-AML2 cells following TAZ knockdown. Data are mean ± SEM of 3 independent experiments. (E) PS:PE ratio in OCI-AML2 cells supplemented with 25 μM PS or vehicle control. (Vehicle control = 1.0.) Data are mean ± SD (n = 4). ***p ≤ 0.001 by Student’s t test. (F) Cell proliferation of OCI-AML2 cells supplemented with 25 μM PS or vehicle control. The relative AUC of viable cell counts over 14 days are shown. Data are relative mean ± SD of a representative experiment from 3 independent experiments (Vehicle control = 100%.) **p ≤ 0.01 by Student’s t test. (G) Clonogenic growth of OCI-AML2 cells pre-treated with 25 μM PS or vehicle control before being seeded in methylcellulose medium without PS. Data are relative mean ± SD of a representative experiment from 3 independent experiments (Vehicle control = 100%.) *p ≤ 0.05 by Student’s t test. (H) Clonogenic growth of primary AML cells pre-treated with 25 μM PS or vehicle control before seeded in methylcellulose medium without PS. Data are relative mean ± SD (n = 2, 0 μM = 100%). **p ≤ 0.01 by Student’s t test. (I and J) Engraftment of TEX cells (I) or 8227 cells (J) treated with PS (25 μM) in NOD-SCID-GF mice. Bar represents mean. (n = 9–10 mice vehicle control group, n = 9–10 mice). **p ≤ 0.01 by Student’s t test. (K) Engraftment of primary AML cells treated with PS (75 μM) or vehicle controls in NOD-SCID-GF mice. Bar represents mean. (n = 10 mice vehicle control group, n = 10 mice PS group.) ***p ≤ 0.001 by Student’s t test. (L) The role of PS decarboxylase (PISD), where PISD decarboxylates PS to produce PE. (M) Immunoblots measuring recombinant PISD protein bound to lipids. Data from 3 independent experiments are shown. (N–Q) PISD protein expression (N) and PS:PE ratio (O) of Cas9-OCI-AML2 cells after CRISPR-mediated PISD knockout. Data are mean ± SD (n = 4). ***p ≤ 0.001 by Student’s t test. (P) Proliferation of Cas9-OCI-AML2 cells after CRISPR-mediated PISD knockout. The relative AUC of viable cell counts over 15 days are shown. Data are mean ± SD of a representative experiment from three independent experiments (control sgRNA = 100%). **p ≤ 0.001 by Student’s t test. (Q) Clonogenic growth of Cas9-OCI-AML2 cells after CRISPR-mediated PISD knockout. Data are relative mean ± SD of a representative experiment from three independent experiments (control sgRNA = 100%). **p ≤ 0.01 by Student’s t test. See also Figures S4 and S5.

Article Snippet: . . Leukemia Cell Lines Five (TAZ knockdown OCI-AML2, and TEX cells) or nine (PISD knockout CAS9-OCI-AML2 cells) days after transduction cells were plated at equal concentrations (CAS9-OCI-AML2 and OCI-AML2 = 750 cells; TEX cells = 2,000) in duplicate 35mm dishes. (Nunclon, Rochester, USA) to a final volume of 1 mL/dish in MethoCult H4100 media (StemCell Technologies, BC, Canada) supplemented with 30% FCS (CAS9-OCI-AML2 and OCI-AML2) or MethoCult H4100 media (StemCell Technologies, BC, Canada) supplemented with 30% FCS, 20 ng/mLSCF, and 2 ng/mL IL-3 (TEX cells).

Techniques: Knockdown, Control, Western Blot, Recombinant, Expressing, CRISPR, Knock-Out

Journal: Cell stem cell

Article Title: The Mitochondrial Transacylase, Tafazzin, Regulates AML Stemness by Modulating Intracellular Levels of Phospholipids

doi: 10.1016/j.stem.2019.02.020

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: . . Leukemia Cell Lines Five (TAZ knockdown OCI-AML2, and TEX cells) or nine (PISD knockout CAS9-OCI-AML2 cells) days after transduction cells were plated at equal concentrations (CAS9-OCI-AML2 and OCI-AML2 = 750 cells; TEX cells = 2,000) in duplicate 35mm dishes. (Nunclon, Rochester, USA) to a final volume of 1 mL/dish in MethoCult H4100 media (StemCell Technologies, BC, Canada) supplemented with 30% FCS (CAS9-OCI-AML2 and OCI-AML2) or MethoCult H4100 media (StemCell Technologies, BC, Canada) supplemented with 30% FCS, 20 ng/mLSCF, and 2 ng/mL IL-3 (TEX cells).

Techniques: Recombinant, Modification, Western Blot, Binding Assay, XF Assay, Electron Microscopy, Staining, High Performance Thin Layer Chromatography, Reverse Transcription, Plasmid Preparation, Apoptosis Assay, DC Protein Assay, SYBR Green Assay, Gene Expression, shRNA, Control, CRISPR, Software