Journal: Haematologica

Article Title: Akt is activated in chronic lymphocytic leukemia cells and delivers a pro-survival signal: the therapeutic potential of Akt inhibition

doi: 10.3324/haematol.2009.010272

Figure Lengend Snippet: Loss of MCL1 through proteasomal degradation is involved in the apoptosis induced by Akt inhibition. (A) CLL cells were cultured in the presence or absence of A-443654. Phospho-GSK-3α constituted a marker of Akt activity, while MCL1 and BCL2 were measured as relevant pro-survival proteins for CLL. Again, PARP cleavage and FACS analysis were used to examine apoptosis, while total GSK-3α and β-actin were used as loading controls. This is a representative example of five experiments on cells from five different CLL cases. (B) Cells were treated as in (A), except that the result of incubation with the pan-caspase inhibitor Z-VAD.fmk in combination with A-443654 was determined. This is a representative example of three experiments involving three different CLL clones. (C) The effect of the proteasome inhibitor, MG-132, was also analyzed. These are representative findings from four separate experiments involving four different CLL clones. In all the above experiments, the inhibitors were added to the cells 1 h prior to treatment with the Akt inhibitor. (D) The effects of knockdown of Akt1 on cell survival and levels of Mcl-1. Here, 1×107 CLL cells were mixed with 100 μL transfection solution (Amaxa) containing a total of 2 μg of siRNA duplexes or 2 μg of non-specific control siRNA before nucleofection using program X-01. Cells (5×106/mL) were subsequently cultured for 72 h, after which levels of Akt1 were measured by western blotting using an Akt1-isoform-specific antibody, while apoptosis was assessed by the FACS method. β-actin constituted a protein loading control. Reduction of Akt1 was associated with loss of p-GSK-3α and MCL1, while total GSK-3α and BCL2 were unaffected.

Article Snippet: Rabbit polyclonal antibodies to MCL1 and BCL2 and a mouse monoclonal anti-MDM2 (clone SMP14) were from Santa Cruz Biotechnology (Insight Biotechnology, Middlesex, UK).

Techniques: Inhibition, Cell Culture, Marker, Activity Assay, Incubation, Clone Assay, Knockdown, Transfection, Control, Western Blot

Journal: Haematologica

Article Title: Akt is activated in chronic lymphocytic leukemia cells and delivers a pro-survival signal: the therapeutic potential of Akt inhibition

doi: 10.3324/haematol.2009.010272

Figure Lengend Snippet: The pro-apoptotic effect of Akt inhibition involves both the GSK-3/MCL1 and p53 pathways. (A) Western blotting analysis was used to determine the effects on MCL1 and BCL2 of prior incubation of CLL cells for 1 h with a GSK-3 inhibitor (SB216763) followed by culture in the presence or absence of A-443654. This is a representative example of three experiments on cells from three different CLL cases. (B) The effects of incubation (24 h) with A-443654 on p53, phospho-MDM2 and p21 are shown. These two panels are representative of five experiments on cells from five different CLL patients.

Article Snippet: Rabbit polyclonal antibodies to MCL1 and BCL2 and a mouse monoclonal anti-MDM2 (clone SMP14) were from Santa Cruz Biotechnology (Insight Biotechnology, Middlesex, UK).

Techniques: Inhibition, Western Blot, Incubation

Journal: Cell metabolism

Article Title: MYC and MCL1 cooperatively promote chemotherapy-resistant breast cancer stem cells through regulation of mitochondrial oxidative phosphorylation

doi: 10.1016/j.cmet.2017.09.009

Figure Lengend Snippet: (A) Plot of genetic alterations as determined by targeted NGS in tumor DNA. X represents no biopsy was available. (B) ALDH+ cells were sorted and then subjected to intracellular labeling with MYC and MCL1 antibodies. (C) Cells were cultured in adherent conditions (ADH) or as mammospheres (MS) for 7 days. Cell lysates were subjected to immunoblot analysis with the indicated antibodies. (D) Relative levels of MYC and MCL1 protein in lysates from TNBC cell lines and quantified by Image J (*p<0.05). (E) MYC and MCL1 protein levels were plotted against the ratio of CD44:CD24 mRNA in the TNBC cell lines (Pearson’s correlation). (F) Levels of MYC mRNA in breast cancer biopsies before chemotherapy (Pre-T) and after chemotherapy (Post-T) were measured by NanoString analysis (n=17; paired t test, *p<0.005). (G) Left panel: H score of IHC analysis of MCL1 from tumor biopsies before chemotherapy and after chemotherapy (n=7; paired t test, *p<0.05). Right panel: Representative MCL1 IHC. (H) MYC, MCL1 and actin immunoblot analyses of lysates from paclitaxel resistant (PCTR) and parental (PAR) cells. Data are represented as mean ± SD.

Article Snippet: Control, MYC and MCL1 shRNA plasmids were purchased from Santa Cruz Biotechnology.

Techniques: Labeling, Cell Culture, Western Blot

Journal: Cell metabolism

Article Title: MYC and MCL1 cooperatively promote chemotherapy-resistant breast cancer stem cells through regulation of mitochondrial oxidative phosphorylation

doi: 10.1016/j.cmet.2017.09.009

Figure Lengend Snippet: (A, C) SUM159PT and MDA-MB-436 cells were transfected with two different MYC or MCL1 siRNAs (A). MDA-MB-468 cells stably transduced with pINDUCER20-MYC (expression induced by 100 ng/mL DOX) or pLX302-GPF or -MCL1 were subjected to mammosphere assay (C). The indicated cells were seeded in mammosphere assays for 7 days (* p<0.05, **p<0.005); original magnification, x100. (B, D) ALDH+ or CD44hi/CD24low fractions in cells manipulated as in A and C were analyzed by FACS (*p<0.005, **p<0.0005). (E, F) SUM159PT cells stably transduced with MYC or MCL1 shRNA were serially diluted and then injected subcutaneously (s.c.) in the lateral dorsum of athymic female mice for ELDA. Data are represented as mean ± SD.

Article Snippet: Control, MYC and MCL1 shRNA plasmids were purchased from Santa Cruz Biotechnology.

Techniques: Transfection, Stable Transfection, Transduction, Expressing, shRNA, Injection

Journal: Cell metabolism

Article Title: MYC and MCL1 cooperatively promote chemotherapy-resistant breast cancer stem cells through regulation of mitochondrial oxidative phosphorylation

doi: 10.1016/j.cmet.2017.09.009

Figure Lengend Snippet: (A–C) OCRs were determined by Seahorse XFe96 extracellular flux analyzer (A). Cells were stained with Mitotracker Red CMXRos and then were analyzed by flow cytometry (B; *p<0.0005) ROS levels were determined by ROS-Glo (C; *p<0.05, **p<0.005). (D) Cells were transduced with MCL1 siRNA and then prepared for transmission electron microscope imaging. Yellow dots outline each individual mitochondrion. (E) Cells transduced with MCL1 were seeded in mammosphere assays and treated with DMSO or 0.1 μM oligomycin A for 7 days (*p<0.05). (F) Cells were transduced with MCL1 siRNA and then lysed and subjected to LC-MS/MS. TCA cycle metabolites were analyzed as described in Methods (Multiple t-tests, *q<0.05, **q<0.0005). (G–I) OCRs and the proportion of CD44hi/CD24low cells were determined by Seahorse XFe96 extracellular flux analyzer (G) and flow cytometry (H), respectively (*p<0.005, **p<0.0005). Cells were seeded in mammosphere assays for 7 days (I; *p<0.05). (J) Cells were transduced with MCL1 siRNA or treated with VU0659158 for 4 days and then seeded in mammosphere assays for 7 days (*p<0.05, ** p <0.005). Data are represented as mean ± SD.

Article Snippet: Control, MYC and MCL1 shRNA plasmids were purchased from Santa Cruz Biotechnology.

Techniques: Staining, Flow Cytometry, Transduction, Transmission Assay, Microscopy, Imaging, Liquid Chromatography with Mass Spectroscopy

Journal: Cell metabolism

Article Title: MYC and MCL1 cooperatively promote chemotherapy-resistant breast cancer stem cells through regulation of mitochondrial oxidative phosphorylation

doi: 10.1016/j.cmet.2017.09.009

Figure Lengend Snippet: (A, C) Cells were transduced with MYC and/or MCL1. After 4 days, cells were seeded in mammosphere assays for 7 days (*p<0.05, **p<0.005, ***p<0.0005). (B, D) Proportion of ALDH+ or CD44hi/CD24low cells was determined by flow cytometry (*p<0.05, **p<0.005, ***p<0.0005). (E, F) MDA-MB-468 cells transduced with pLX302-GFP or -MCL1 were re-transduced with pINDUCER20-MYC. Cells were then seeded in a mammosphere assay for 7 days ± 100 ng/mL DOX (E). Proportion of CD44hi/CD24low cells was determined by flow cytometry after 4 days of treatment with DOX (F). (G) OCRs were determined by Seahorse XFe96 extracellular flux analyzer. (H) ROS levels were examined by ROS-Glo (*p<0.05, **p<0.005, ***p<0.0005). (I) GSVA score was examined with a gene set [Reactome_Pyruvate metabolism and Citric Acid (TCA) cycle] in breast cancers in TCGA (*p<0.0005). Data are represented as mean ± SD.

Article Snippet: Control, MYC and MCL1 shRNA plasmids were purchased from Santa Cruz Biotechnology.

Techniques: Transduction, Flow Cytometry