Journal: Cell Death & Disease

Article Title: Oxidized ATM promotes breast cancer stem cell enrichment through energy metabolism reprogram-mediated acetyl-CoA accumulation

doi: 10.1038/s41419-020-2714-7

Figure Lengend Snippet: a Hs578T and BT549 cells were exposed to 21 or 1% O 2 for 24 h; CD44 + /CD24 − cells in both Hs578T and BT549 cell populations were detected by flow cytometry (* P < 0.05). b Representative images of mammospheres formed from Hs578T and BT549 cells in 1% O 2 condition (scale bar, 100 µm). The quantification of mammosphere formation efficiencies (sphere number) and the mean sphere sizes was shown (** P < 0.01). c , d qRT-PCR ( c ) and western blotting ( d ) were employed to determine the expressions of the indicated CSC-associated genes in mammospheres of Hs578T and BT549 cells under hypoxia conditions (** P < 0.01).

Article Snippet: Human breast cancer cells Hs578T and BT549 were obtained from ATCC (American Type Culture Collection).

Techniques: Flow Cytometry, Quantitative RT-PCR, Western Blot

Journal: Cell Death & Disease

Article Title: Oxidized ATM promotes breast cancer stem cell enrichment through energy metabolism reprogram-mediated acetyl-CoA accumulation

doi: 10.1038/s41419-020-2714-7

Figure Lengend Snippet: Hs578T and BT549 cells, and ATM-silenced Hs578T and BT549 cells by transfectting two different shRNAs against ATM (shATM#1 and shATM#2) were suspensded in culture in CSC medium under normaxia (21% O 2 ) or hypoxia (1% O 2 ) and treated with or without ATM inhibitor KU60019 (ATM inhibitor, 10 µM). a The representative images of mammospheres are shown (scale bar, 100 µm). b The quantification of mammosphere formation efficiency. Data are shown as the mammospheres number/1000 seeded cells ± SEM from three independent experiments (** P < 0.01) (Nor normoxia, Hyp hypoxia). c The quantification of mammosphere sizes. The data represent means ± SD from three independent experiments (* P < 0.01, hypoxia vs normoxia; ** P < 0.01, vs hypoxic control). d The representative images of mammospheres formed from breast tumor with low oxidized ATM and high oxidized ATM (scale bar, 100 µm) under normoxia or hypoxia condition and treatment with or without KU60019 (10 μM). e The mammosphere sphere number and mean sphere sizes of d were quantified (* P < 0.05, ** P < 0.01).

Article Snippet: Human breast cancer cells Hs578T and BT549 were obtained from ATCC (American Type Culture Collection).

Techniques: Control

Journal: Cell Death & Disease

Article Title: Oxidized ATM promotes breast cancer stem cell enrichment through energy metabolism reprogram-mediated acetyl-CoA accumulation

doi: 10.1038/s41419-020-2714-7

Figure Lengend Snippet: a Metabonomics analysis was performed to identify the changed metabolites between oxidized ATM-activated CSC and -inactivated CSC derived from Hs578T under hypoxia, and relative metabolic signaling pathways were enriched by KEGG analysis. Ratio represented the number of changed metabolites to the total number of metabolites in the pathway. b – i Hs578T and BT549 cells, and ATM-silenced Hs578T and BT549 cells were suspended in culture in CSC medium under hypoxia and treated with or without indicated reagents (e.g., 2-deoxyglucose (2-DG, 5 mM), metformin (3 mM), and/or KU60019 (10 µM)). The relative of ATP products ( b ) and metabolic index were checked. Glucose consumption of mammospheres ( c ); the productions of pyruvate ( d ), lactate ( e ), acetyl-CoA ( f ), citrate ( g ), succinate ( h ), and fumarate ( i ) were shown. j CSC incorporation of 13 C 6 -labeled glucose was added into CSC medium and pulsed for up to 8 h; the isotopolog distribution for pyruvate, citrate, succinate, and fumarate with two 13 C atoms in sphere cells was shown. k – m CSCs were treated with or without BMS303141 (an inhibitor of ATP-citrate lyase, 20 μM); lactate, succinate, cytosolic, and mitochondrial acetyl-CoA concentrations were determined (* P < 0.05, ** P < 0.01).

Article Snippet: Human breast cancer cells Hs578T and BT549 were obtained from ATCC (American Type Culture Collection).

Techniques: Derivative Assay, Protein-Protein interactions, Labeling

Journal: Cell Death & Disease

Article Title: Oxidized ATM promotes breast cancer stem cell enrichment through energy metabolism reprogram-mediated acetyl-CoA accumulation

doi: 10.1038/s41419-020-2714-7

Figure Lengend Snippet: a , b Western blotting show the glycolysis-related ( a ) and TCA-cycle-related ( b ) gene expressions in mammospheres derived from Hs578T and BT549 cells under treatment with or without KU60019 (10 µM) and from ATM-silenced cells in hypoxia. c – h Acetyl-CoA productions of the indicated mammospheres were determined. c Knockdown of GLUT1, PKM2, or PDHa and the control mammospheres. d Ectopic GLUT1, PKM2, or PDHa was transfected into hypoxic mammospheres with ATM knocked down. e Mammospheres treated with or without 2-deoxyglucose (2-DG, 5 mM), UK5099 (an inhibitor of the mitochondrial pyruvate carrier (MPC), 10 μM), or BMS303141 (an inhibitor of ACLY, 20 μM); f mammospheres treated with or without Etomoxir (ETO, an inhibitor of CPT1, 20 μM); g exogenous addition with or without acetate (1, 5, or 10 mM). h Exogenous addition with or without acetate (10 mM) in mammospheres under ATM, GLUT1, PKM2, or PDHa knocked down. i , j A positive correlation between p-ATM and SOX2 protein levels ( i ), p-ATM and production of acetyl-CoA ( j ), and SOX2 protein expression and acetyl-CoA ( k ) in 92 clinical breast cancer samples (* P < 0.05; ** P < 0.01).

Article Snippet: Human breast cancer cells Hs578T and BT549 were obtained from ATCC (American Type Culture Collection).

Techniques: Western Blot, Derivative Assay, Knockdown, Control, Transfection, Expressing

Journal: Cell Death & Disease

Article Title: Oxidized ATM promotes breast cancer stem cell enrichment through energy metabolism reprogram-mediated acetyl-CoA accumulation

doi: 10.1038/s41419-020-2714-7

Figure Lengend Snippet: a Protein levels of acetylated-lysine histone H3 and H4 were determined by immunoblotting in cell lysates from Hs578T and BT549 spheres treated with or without KU60019 (10 μM) under hypoxia. β-Actin is the loading control. b Immunofluorescence staining to check acetylated-H4ac levels in Hs578T and BT549 mammospheres treated with or without KU60019 (scale bar, 100 µm) under hypoxia. c – e The status of H4 acetylation was determined in hypoxic Hs578T and/or BT549 mammospheres suffered to different treatments. c Spheres treated with KU60019 (10 μM); d those treated with 2-DG (5 mM), UK5099 (15 μM), and BMS303141 (20 μM), respectively; e those treated with acetate (10 mM). Total H4 is the loading control.

Article Snippet: Human breast cancer cells Hs578T and BT549 were obtained from ATCC (American Type Culture Collection).

Techniques: Western Blot, Control, Immunofluorescence, Staining

Journal: Cell Death & Disease

Article Title: Oxidized ATM promotes breast cancer stem cell enrichment through energy metabolism reprogram-mediated acetyl-CoA accumulation

doi: 10.1038/s41419-020-2714-7

Figure Lengend Snippet: a Photos of the xenograft tumors dissected from the nude mice at the seventh week after being subcutaneously injected with BT549 mammosphere cells. When tumor volume reaches around 50 mm 3 , mice were randomized into five groups and administered with KU60019 (50 mg/kg, three times a week), or UK5099 (10 μg/kg, once every 2 days), acetate (150 mg/kg, three times a week), and acetate (250 mg/kg, three times a week) alone or in combination, via intraperitoneal (i.p.) injection. b Tumor growth curve (* P < 0.05, ** P < 0.01). c The average tumor weight of each group. d Acetyl-CoA productions of xenograft tumors. e GLUT1, PKM2, PDHa, H4ac, and SOX2 levels were determined in tumor tissues by IHC analyses.

Article Snippet: Human breast cancer cells Hs578T and BT549 were obtained from ATCC (American Type Culture Collection).

Techniques: Injection

Journal: The EMBO Journal

Article Title: Reciprocal priming between receptor tyrosine kinases at recycling endosomes orchestrates cellular signalling outputs

doi: 10.15252/embj.2020107182

Figure Lengend Snippet:

Article Snippet: BT549 , LGC ATCC® , HTB‐122.

Techniques: Virus, Recombinant, Extraction, Multiplex sample analysis, Transfection, Molecular Weight, Marker, Reverse Transcription, Protease Inhibitor, Membrane, Cell Culture, Clinical Proteomics, Spectroscopy, Imaging, cDNA Synthesis, Plasmid Preparation, Negative Control, Mutagenesis, Clone Assay, Generated, Software, Microscopy

Journal: Molecular Therapy Oncolytics

Article Title: Upregulation of endogenous TRAIL-elicited apoptosis is essential for metformin-mediated antitumor activity against TNBC and NSCLC

doi: 10.1016/j.omto.2021.04.012

Figure Lengend Snippet: Metformin induced apoptosis in both TNBC and NSCLC cells (A) TNBC cells (HCC70, MDA-MB-468, and BT549) and NSCLC cells (H460, H1650, and A549) were plated onto 6 cm dishes with culture medium containing 10% FBS. After 24 h, cells were treated with indicated concentrations of metformin in fresh medium with 5% FBS for 48 h. Both adherent and non-adherent cells were collected and subjected to apoptosis analysis using a cell death detection ELISA. Data show a representative of three independent experiments. Bars, SD. ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. (B) The same batch of cell lysates was used for western blot analyses with specific antibodies directed against PARP, caspase-8, caspase-3, or β-actin.

Article Snippet: Human TNBC cell lines (HCC70, MDA-MB-468, MDA-MB-231, and BT549) and NSCLC cell lines (H460, H1650, and A549) were from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Molecular Therapy Oncolytics

Article Title: Upregulation of endogenous TRAIL-elicited apoptosis is essential for metformin-mediated antitumor activity against TNBC and NSCLC

doi: 10.1016/j.omto.2021.04.012

Figure Lengend Snippet: Metformin upregulated TRAIL protein levels without altering expression of DR4 and DR5 in TNBC and NSCLC cells (A) TNBC and NSCLC cells were treated with different concentrations of metformin in 5% FBS medium for 48 h. Cells were collected and subjected to western blot analysis of TRAIL expression. The densitometry analyses of TRAIL signals were shown underneath, and the arbitrary numbers indicated the intensities of each cell line relative to controls, defined as 1.0. (B) Western blot analysis of TRAIL receptors DR5 and DR4 expression in NSCLC and TNBC cells treated with metformin for 48 h. (C) Flow cytometric analysis of membrane DR5 and DR4 in NSCLC and TNBC cells with or without metformin treatment. Cells were stained with APC-conjugated DR5 and PE-conjugated DR4 antibodies (tinted histograms) or with the isotype-matched IgG control (shadowed histogram). Data show a representative of three independent experiments with similar results. (D) The mean fluorescent intensity (MFI) of DR4 and DR5 was quantified. Values were expressed as the mean ± SD of three independent experiments. ns, not significant

Article Snippet: Human TNBC cell lines (HCC70, MDA-MB-468, MDA-MB-231, and BT549) and NSCLC cell lines (H460, H1650, and A549) were from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Western Blot, Membrane, Staining, Control

Journal: Molecular Therapy Oncolytics

Article Title: Upregulation of endogenous TRAIL-elicited apoptosis is essential for metformin-mediated antitumor activity against TNBC and NSCLC

doi: 10.1016/j.omto.2021.04.012

Figure Lengend Snippet: Metformin promoted TRAIL secretion by TNBC and NSCLC cells (A) Schematic representation of the quantitative and functional analyses of the soluble TRAIL in the conditioned medium (CM). In brief, 1 × 10 6 H460 or MDA-MB-468 cells were plated onto 10 cm dishes with medium containing 10% FBS. After 24 h, the culture medium was replaced with fresh medium containing 0.5% FBS as control or the same medium containing metformin. After 48 h incubation, the cells’ CM was collected and concentrated 50-fold with the Millipore centrifugal concentration column. The concentrated CM was subjected to quantitative analysis and bioactivity assays. (B) The levels of soluble TRAIL in the concentrated CM were determined with western blots and a TRAIL-specific ELISA. Data show a representative of three independent experiments. Bars, SD. ND, not detected. (C and D) MDA-MB-231 cells were incubated with the conditioned CM of H460 cells in the absence or presence of the TRAIL-R2 Fc chimera protein (200 ng/mL) for 24 h. MDA-MB-231 cells incubated with fresh medium with or without metformin were used as controls. After 24 h, MDA-MB-231 cells were subjected to LIVE/DEAD Cell Imaging. Green, live cells; red, dead cells. The ratio of dead/live cells was determined by using the number of dead cells (red) dividing the number of live cells (green) in each sample (C). The same batch of cells was subjected to apoptosis ELISA (D). Bars, SD.

Article Snippet: Human TNBC cell lines (HCC70, MDA-MB-468, MDA-MB-231, and BT549) and NSCLC cell lines (H460, H1650, and A549) were from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Functional Assay, Control, Incubation, Concentration Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Imaging

Journal: Molecular Therapy Oncolytics

Article Title: Upregulation of endogenous TRAIL-elicited apoptosis is essential for metformin-mediated antitumor activity against TNBC and NSCLC

doi: 10.1016/j.omto.2021.04.012

Figure Lengend Snippet: Inhibition of TRAIL function with a recombinant TRAIL-R2 Fc chimera significantly attenuated metformin-induced apoptosis in TNBC and NSCLC cells (A) TNBC and NSCLC cells were plated onto 6 cm dishes with medium containing 10% FBS. After 24 h, the culture medium was replaced with fresh medium with 5% FBS containing either metformin or TRAIL-R2 Fc chimera protein alone or combinations of metformin and TRAIL-R2 Fc chimera for 48 h. Both adherent and non-adherent cells were collected and subjected to apoptosis ELISA. Data show a representative of three independent experiments. Bars, SD. ns, not significant, ∗p < 0.05, ∗∗p < 0.01. (B) The same batch of cell lysates was used for western blot analyses with specific antibodies directed against PARP, caspase-8, caspase-3, or β-actin.

Article Snippet: Human TNBC cell lines (HCC70, MDA-MB-468, MDA-MB-231, and BT549) and NSCLC cell lines (H460, H1650, and A549) were from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Inhibition, Recombinant, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Molecular Therapy Oncolytics

Article Title: Upregulation of endogenous TRAIL-elicited apoptosis is essential for metformin-mediated antitumor activity against TNBC and NSCLC

doi: 10.1016/j.omto.2021.04.012

Figure Lengend Snippet: Specific knockdown of TRAIL expression inhibited metformin-induced apoptosis in TNBC and NSCLC cells TNBC and NSCLC cells were infected with lentivirus containing either control shRNA (sh-scr) or specific TRAIL -targeting shRNA (sh-1 or sh-2). The infected cells were selected by puromycin for 24 h and then treated with or without metformin for additional 48 h. (A) Both adherent and non-adherent cells were collected and subjected to western blot analyses of TRAIL, PARP, caspase-8, caspase-3, or β-actin. The densitometry analyses of TRAIL signals were shown underneath, and the arbitrary numbers indicated the intensities of each cell line relative to controls, defined as 1.0. (B) The same batch of cell lysates was subjected to apoptosis ELISA. Data show a representative of three independent experiments. Bars, SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.01, ∗∗∗∗p < 0.001.

Article Snippet: Human TNBC cell lines (HCC70, MDA-MB-468, MDA-MB-231, and BT549) and NSCLC cell lines (H460, H1650, and A549) were from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Knockdown, Expressing, Infection, Control, shRNA, Western Blot, Enzyme-linked Immunosorbent Assay