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

Journal: Nature Communications

Article Title: Targeting anti-apoptotic pathways eliminates senescent melanocytes and leads to nevi regression

doi: 10.1038/s41467-022-35657-9

Figure Lengend Snippet: a Protein expression and densitometry analysis of MCL-1 in irradiated melanocytes treated with 5 µM ABT-263 after 6, 12, or 24 h. Corrected total cell fluorescence of MCL-1 in senescent b irradiated and c BRAF V600E expressing melanocytes treated for 24 h with DMSO or ABT-263. d Cycloheximide chase experiment of MCL-1 in DMSO and ABT-263 treated irradiated melanocytes. Cells were incubated with DMSO or 5 µM ABT-263 for 12 h, after which 5 µg/ml cycloheximide was added. Protein lysates were obtained after the indicated time points and used for Mcl-1 immunoblotting. Quantification of MCL-1 degradation rates was carried out using densitometry analysis with vinculin as a housekeeping protein. Expression was normalized to protein levels at time point 0. e Distribution of Mcl-1 mRNA in monosome, light polysome, or heavy polysome fractions from irradiated melanocytes treated with DMSO or 5 µM ABT-263 for 16 h. Distribution of β-actin mRNA was used to normalize values. f Immunoblot of immunoprecipitated MCL-1 in vehicle or ABT-263 treated irradiated melanocytes showing nascent (biotinylated) MCL-1 synthesis. NBT nitro-blue-tetrazalium. Ratio of biotinylated (nascent) MCL-1 protein to total MCL-1 protein in MCL-1 IP fraction. g Protein expression of MCL-1 in irradiated melanocytes treated with DMSO, 5 µM ABT-263, and/or 50 µM 4EGI-1. h Viability of non-irradiated and irradiated melanocytes treated with 5 µM ABT-263 and/or 50 µM 4EGI-1. i Protein expression and densitometry analysis of MCL-1 in DMSO or 1 µM PP242 treated irradiated melanocytes after 24 h. Vinculin was used as a housekeeping protein and expression was normalized to protein levels in DMSO treated cells. j Quantification of Annexin V/Pi flow cytometric analysis of non-senescent (NS) and senescent (Sen IR) melanocytes treated with DMSO, 5 µM ABT-263, 1 µM PP242, or 2.5 µM ABT-263/1 µM PP242 for 24 h (see supplementary fig. ). The percentage of viable cells in drug-treated cells were normalized to the percentage of DMSO treated viable cells. k Annexin V/Pi flow cytometric analysis and quantification of EV or plx307-Mcl1 transduced irradiated melanocytes treated with 2.5 µM ABT-263/1 µM PP242 for 24 h. For all panels significance was calculated with a two-tailed student’s t test and n = 3 independent experiments except k where n = 4. Error bars = S.E.M. * p < 0.05, ** p < 0.01, *** p < 0.001, ns = not significant.

Article Snippet: Mcl-1 overexpression was carried out using the plx307-MCL1 plasmid (Addgene # 117726).

Techniques: Expressing, Irradiation, Fluorescence, Incubation, Western Blot, Immunoprecipitation, Two Tailed Test