Journal: Nature Communications

Article Title: Essentiality of fatty acid synthase in the 2D to anchorage-independent growth transition in transforming cells

doi: 10.1038/s41467-019-13028-1

Figure Lengend Snippet: FASN is essential for PyMT-dependent transformation in vitro. a Representative images of the soft agar colony assay and quantitation of colonies per well recovered from each of the four genotypes ( n = 6 biological replicates for each genotype). Scale bars, 500 μM. b FASN mRNA and protein levels detected in soft agar colonies recovered from the FASN lox/lox -PyMT and FASN ∆/∆ -PyMT MEFs, respectively. c Cell replication rates after 1, 2, or 3 days for each genotype ( n = 3). Presented data are the mean values ± standard deviation (SD). *** P < 0.001; n.s., not statistically significant; Student’s t test. d Flow cytometry analysis of apoptosis. Percentages of cells in apoptosis are represented (FASN lox/lox -Empty, n = 7; FASN ∆/∆ -Empty, n = 8; FASN lox/lox -PyMT, n = 7; FASN ∆/∆ -PyMT, n = 5). Presented data are the mean values ± standard deviation (SD)

Article Snippet: The following primary Abs were used: FASN (1:1000, clone: C20G5; 3180, Cell Signaling), PyMT (1:2500, NB100-2749, Novus Biological), K-Ras (1:1000, clone: F234; sc-30; Santa Cruz), HER2 (1:1000, 2242, Cell Signaling), IDH1 (1:1000, clone: D2H1, 8137, Cell Signaling), β-actin (1:5000, clone AC-15; A5441, Sigma), γ-tubulin (1:2500, clone GTU-88; T6557, Sigma), and vinculin (1:2000, clone: hVIN-1; V9131, Sigma).

Techniques: Transformation Assay, In Vitro, Colony Assay, Quantitation Assay, Standard Deviation, Flow Cytometry

Journal: Nature Communications

Article Title: Essentiality of fatty acid synthase in the 2D to anchorage-independent growth transition in transforming cells

doi: 10.1038/s41467-019-13028-1

Figure Lengend Snippet: FASN deficit impairs the upregulation of glycolysis and the anaplerotic shift of Krebs cycle. a The lack of FASN led to an accumulation of NADPH, acetyl-, and malonyl-CoA. The increase in acetyl-CoA can allosterically inhibit the activity of pyruvate dehydrogenase. This can block the entry of pyruvate into the Krebs cycle (associated with a decrease in the NAD-NADH cycling, respiration, and glycolytic rates) and the derivation of carbon skeletons to biosynthetic routes. In addition, a decrease in Krebs cycle activity could be coupled to a limited ability to satisfy high energetic demands. Pyruvate accumulation could also decrease glycolysis, which in addition to limiting ATP production would impair the ability to derive carbon skeletons to the nucleotide synthesis pathways. All these processes are upregulated in transformed compared with normal cells and are a hallmark of cancer . b Total abundance of acetyl-CoA and NADPH determined by LC/MS across the four genotypes ( n = 4). c Representative extracellular acidification rate (ECAR) measurements. Glucose, oligomycin, and 2-deoxyglucose (2-DG) were added at the indicated time point for each experiment. d Mitochondrial respiration reflected by oxygen consumption rate levels (OCR) was detected in the four genotypes. In c , d presented data are mean values ± SEM. e Mitotracker dye (green) was used to reveal the localization of mitochondria (left), and the positively charged dye TMRE (red) (middle) was used to compare mitochondrial transmembrane polarization. Representative fluorescence images of TMRE and Mitotracker were taken by confocal microscopy. Merged images are shown. Scale bars, 10 μM. Quantitative analysis of relative fluorescence intensity is shown in the right-hand side (FASN lox/lox -Empty, n = 388 cells; FASN ∆/∆ -Empty, n = 458 cells; FASN lox/lox -PyMT, n = 294 cells; FASN ∆/∆ -PyMT, n = 382 cells). FCCP was added to FASN lox/lox -PyMT as a negative control ( n = 90 cells). f Representative flow cytometry analysis (left) showing constitutive mitochondrial superoxide levels for each genotype ( n = 4). The quantitation of MitoSOX red fluorescence intensity is shown in the right. In b , e , and f represented data are the mean values ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001; Student’s t test

Article Snippet: The following primary Abs were used: FASN (1:1000, clone: C20G5; 3180, Cell Signaling), PyMT (1:2500, NB100-2749, Novus Biological), K-Ras (1:1000, clone: F234; sc-30; Santa Cruz), HER2 (1:1000, 2242, Cell Signaling), IDH1 (1:1000, clone: D2H1, 8137, Cell Signaling), β-actin (1:5000, clone AC-15; A5441, Sigma), γ-tubulin (1:2500, clone GTU-88; T6557, Sigma), and vinculin (1:2000, clone: hVIN-1; V9131, Sigma).

Techniques: Activity Assay, Blocking Assay, Transformation Assay, Liquid Chromatography with Mass Spectroscopy, Fluorescence, Confocal Microscopy, Negative Control, Flow Cytometry, Quantitation Assay

Journal: Nature Communications

Article Title: Essentiality of fatty acid synthase in the 2D to anchorage-independent growth transition in transforming cells

doi: 10.1038/s41467-019-13028-1

Figure Lengend Snippet: Decreased glucose carbon flux and the pentose phosphate pathway. a Schematic representing incorporation of 13 C derived from glucose into metabolites of glycolysis and the Krebs cycle. White and black circles are 12 C and 13 C, respectively. b Decreased labeled but similar total palmitate pools in FASN ∆/∆ -PyMT MEFs compared with FASN lox/lox -PyMT MEFs. 13 C-NMR spectrum showing the central peak (singlet) at 14.17 p.p.m. characteristic of the terminal methyl group (–CH 3 or omega-1 carbon) of a fatty acid, and lateral peaks (doublet signal) corresponding to the penultimate –CH 2 - (or omega-2 carbon) due to 13 C– 13 C coupling. c Isotopolog abundance of glucose-6-P (determined by LC/MS) and lactate (determined by RMN). d Glut1 and Glut4 glucose transporter mRNA levels determined by RT-qPCR, relativized to FASN lox/lox -PyMT values. The experiment was repeated three times. e Total abundance of citrate/isocitrate and % labeling of citrate, aconitate, fumarate, and malate isotopologs in MEFs labeled with [U- 13 C]glucose (determined by LC/MS) ( n = 5 biological replicates), and f % labeling of –CH 2 succinate isotopomer (determined by RMN) ( n = 5 biological replicates). g Isotopolog abundance of ADP ribose and AMP determined by LC/MS in FASN lox/lox -PyMT and FASN ∆/∆ -PyMT MEFs ( n = 5 biological replicates). Presented data are the mean values ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001; n.s., not statistically significant; Student’s t test

Article Snippet: The following primary Abs were used: FASN (1:1000, clone: C20G5; 3180, Cell Signaling), PyMT (1:2500, NB100-2749, Novus Biological), K-Ras (1:1000, clone: F234; sc-30; Santa Cruz), HER2 (1:1000, 2242, Cell Signaling), IDH1 (1:1000, clone: D2H1, 8137, Cell Signaling), β-actin (1:5000, clone AC-15; A5441, Sigma), γ-tubulin (1:2500, clone GTU-88; T6557, Sigma), and vinculin (1:2000, clone: hVIN-1; V9131, Sigma).

Techniques: Derivative Assay, Labeling, Liquid Chromatography with Mass Spectroscopy, Quantitative RT-PCR

Journal: Nature Communications

Article Title: Essentiality of fatty acid synthase in the 2D to anchorage-independent growth transition in transforming cells

doi: 10.1038/s41467-019-13028-1

Figure Lengend Snippet: FASN deletion leads to ATP citrate lyase (ACLY) inhibition, blocking 3D growth. a Representative pictures of the spheroids recovered from the different genotypes growing under ultralow attachment conditions for 72 h (left). Spheroid diameter ( n = 30 spheroids, FASN lox/lox -PyMT; n = 24 FASN ∆/∆ -PyMT) and number of cells over 72 h (right). Scale bars, 500 μM. Presented data are the mean values ± SD. *** P < 0.001; Student’s t test. b OCR assay performed with cells recovered from the previous experiment and monolayer cultures. Oligomycin, FCCP, and antimycin/rotenone were added at the indicated time point. Data are represented as the means ± SEM. c Representative flow cytometry analysis showing constitutive mitochondrial superoxide levels in monolayer compared with 3D conditions in PyMT MEFs loaded with MitoSOX Red (FASN lox/lox -PyMT, n = 4; FASN ∆/∆ -PyMT, n = 5). The quantification of MitoSOX Red fluorescence intensity is shown on the left. Presented data are the mean values ± SD. *** P < 0.001, Student’s t test. d Quantification of MitoSOX Red fluorescence intensity by flow cytometry analysis in FASN lox/lox -PyMT, FASN ∆/∆ -PyMT, and DMNQ-treated (10 μM; 20 min) FASN lox/lox -PyMT MEFs ( n = 3). e Effect of 10 μM DMNQ treatment in the ability of FASN lox/lox -PyMT MEFs to form spheroids. Scale bars, 500 μM. Representative pictures for each condition are shown. f Representative pictures showing the effects of sodium citrate (5 mM) or SB204990 (30 μM) in the formation of tumor spheroids by FASN lox/lox -PyMT MEFs. Scale bars, 500 μM. The lower charts show an increase in ROS production by sodium citrate and SB204990. Representative flow cytometry analysis was shown ( n = 4). g Effects in OCR suppression caused by sodium citrate or SB204990 treatment in FASN lox/lox -PyMT MEFs. Presented data are the mean values ± SEM. Three independent experiments ( h ) ACLY activity in FASN lox/lox -PyMT versus FASN ∆/∆ -PyMT MEFs. i , j Cell viability was determined in FASN lox/lox -PyMT MEFs at 24, 48, and 72 h in the presence of various concentrations of SB204990 ( n = 8) ( i ) or sodium citrate ( n = 7) ( j ) under 2D culture conditions. In h – j represented data are the mean values ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001; Student’s t test

Article Snippet: The following primary Abs were used: FASN (1:1000, clone: C20G5; 3180, Cell Signaling), PyMT (1:2500, NB100-2749, Novus Biological), K-Ras (1:1000, clone: F234; sc-30; Santa Cruz), HER2 (1:1000, 2242, Cell Signaling), IDH1 (1:1000, clone: D2H1, 8137, Cell Signaling), β-actin (1:5000, clone AC-15; A5441, Sigma), γ-tubulin (1:2500, clone GTU-88; T6557, Sigma), and vinculin (1:2000, clone: hVIN-1; V9131, Sigma).

Techniques: Inhibition, Blocking Assay, Flow Cytometry, Fluorescence, Activity Assay

Journal: Nature Communications

Article Title: Essentiality of fatty acid synthase in the 2D to anchorage-independent growth transition in transforming cells

doi: 10.1038/s41467-019-13028-1

Figure Lengend Snippet: FASN deficit leads to an impairment of IDH1-dependent reductive carboxylation. a Isotopolog abundance of α-ketoglutarate, b fumarate, and c and citrate/isocitrate from [U- 13 C 5 ]glutamine in 2D cultures determined by LC/MS ( n = 4 biological replicates). d Isotopolog abundance of α-ketoglutarate, e fumarate, and f citrate/isocitrate from [U- 13 C 5 ]glutamine in 3D cultures determined by LC/MS ( n = 4 biological replicates). Presented data are the mean values ± SD. * P < 0.05; Student’s t test. g Relative levels of NADPH in cellular and mitochondrial lysates of FASN lox/lox -PyMT versus FASN ∆/∆ -PyMT MEFs in 3D cultures ( n = 3). Presented data are the mean values ± SD. * P < 0.05 and *** P < 0.001; Student’s t test. h Digitonin-solubilized isolated mitochondria from FASN lox/lox -PyMT and FASN ∆/∆ -PyMT MEFs in 2D and 3D culture conditions were subjected to blue native gel electrophoresis (BNGE) followed by in-gel complex I activity assay. Complex I occurs both free and bound with complex III (I + III 2 and I 2 + III 2 supercomplexes). Relative abundance of assembled complex I into supercomplexes (SC) versus free complex I (CI) after the quantification of band intensity in direct immunobloting of BNGEC against complex I subunit NDUFS1 in FASN lox/lox -PyMT versus FASN ∆/∆ -PyMT in 2D and 3D culture conditions is shown. Presented data are the mean values ± SD ( n = 2 independent culture preparation). * P < 0.05; Student’s t test. i NAC (1 mM) and GSH-MEE (1 mM) rescue experiments. Data are represented as mean values ± SEM. Three independent experiments. j Rescue of soft-agar colonies (above) and spheroids (below) in FASN ∆/∆ -PyMT MEFs treated with NAC (1 mM) or GSH-MEE (1 mM). Representative images for each condition are shown. Scale bars, 500 μM. k Representative flow cytometry analysis showing constitutive mitochondrial superoxide levels in FASN ∆/∆ -PyMT MEFs treated with NAC or GSH-MEE ( n = 4). MitoSOX red fluorescence intensity quantitation is shown on the right. Presented data are the mean values ± SD. ** P < 0.01; Student’s t test. l NAD + /NADH ratio is restored after the addition of NAC or GSH-MEE in FASN ∆/∆ -PyMT MEFs in 3D cultures ( n = 4). Presented data are the mean values ± SD. * P < 0.05; Student’s t test

Article Snippet: The following primary Abs were used: FASN (1:1000, clone: C20G5; 3180, Cell Signaling), PyMT (1:2500, NB100-2749, Novus Biological), K-Ras (1:1000, clone: F234; sc-30; Santa Cruz), HER2 (1:1000, 2242, Cell Signaling), IDH1 (1:1000, clone: D2H1, 8137, Cell Signaling), β-actin (1:5000, clone AC-15; A5441, Sigma), γ-tubulin (1:2500, clone GTU-88; T6557, Sigma), and vinculin (1:2000, clone: hVIN-1; V9131, Sigma).

Techniques: Liquid Chromatography with Mass Spectroscopy, Isolation, Nucleic Acid Electrophoresis, Activity Assay, Western Blot, Flow Cytometry, Fluorescence, Quantitation Assay

Journal: Nature Communications

Article Title: Essentiality of fatty acid synthase in the 2D to anchorage-independent growth transition in transforming cells

doi: 10.1038/s41467-019-13028-1

Figure Lengend Snippet: fects of FASN deletion in tumor development. a FASN-stained slides from wild-type and MMTV-PyMT FVB mice mammary pads at weeks 4, 8, and 13. FASN is expressed in the cytoplasm of adipocytes (asterisks) in wild-type animals. A weaker FASN expression was observed in breast epithelial ducts (arrows). Conversely, FASN upregulation was observed in the proliferating ducts (week 4), invasive carcinomas arising from the ducts (week 8), and fully developed tumors (week 13) in the MMTV-PyMT animals. Scale bars, 100 μM. b Representative images of sections of the mammary pad of FASN +/+ ; PyMT and FASN ∆/∆ ; PyMT animals stained for FASN. The areas negative for FASN have normal or at most proliferative (but not invasive) ducts (asterisks). Invasive growth was only observed in the FASN-positive areas of the FASN ∆/∆ ; PyMT mice (arrows). Scale bars, 200 μm (low magnification) and 100 μm (high magnification; i, ii, iii). c Representative fluorescent images of DAPI (blue) and 8-oxo-deoxyguanosine (8-oxo-dG) (red) staining of mammary pad sections of FASN +/+ ; PyMT and FASN ∆/∆ ; PyMT animals. Merged images are shown. The quantification of cellular 8-oxo-dG fluorescence intensity in breast tumors is shown on the right (FASN +/+ ; PyMT, n = 4500 cells; FASN ∆/∆ ; PyMT, n = 6500 cells). Presented data are the mean values ± SD. *** P < 0.001; Student’s t test. Scale bars, 50 μM. d Representative fluorescent images of DAPI (blue), FASN (green), and 8-oxo-dG (red) co-staining of mammary pad section of FASN ∆/∆ ; PyMT animals. Scale bars, 50 μM. e Time to killing due to loco-regional tumor growth of FASN +/+ ; PyMT ( n = 43) and FASN ∆/∆ ; PyMT ( n = 44) animals; survival is increased from 90 to 123 days (log-rank P < 0.0001). f Tumor volume evolution in FASN +/+ ; PyMT ( n = 43) and FASN ∆/∆ ; PyMT ( n = 44) animals. Representative tumor burden charts at T4, T5, and T6 weeks (since treatment initiation) are shown below the survival curve (T4: FASN +/+ ; PyMT n = 42, FASN ∆/∆ ; PyMT n = 16; T5: FASN +/+ ; PyMT n = 41, FASN ∆/∆; PyMT n = 15; T6: FASN +/+ ; PyMT n = 40, FASN ∆/∆; PyMT n = 15). Presented data are the mean values ± SEM. *** P < 0.001; Student’s t test

Article Snippet: The following primary Abs were used: FASN (1:1000, clone: C20G5; 3180, Cell Signaling), PyMT (1:2500, NB100-2749, Novus Biological), K-Ras (1:1000, clone: F234; sc-30; Santa Cruz), HER2 (1:1000, 2242, Cell Signaling), IDH1 (1:1000, clone: D2H1, 8137, Cell Signaling), β-actin (1:5000, clone AC-15; A5441, Sigma), γ-tubulin (1:2500, clone GTU-88; T6557, Sigma), and vinculin (1:2000, clone: hVIN-1; V9131, Sigma).

Techniques: Staining, Expressing, Fluorescence

Journal: Nature Communications

Article Title: Essentiality of fatty acid synthase in the 2D to anchorage-independent growth transition in transforming cells

doi: 10.1038/s41467-019-13028-1

Figure Lengend Snippet: FASN inhibition prevents the development of invasive ductal carcinoma. a Inhibitory effects of G28UCM on tumor growth in MMTV-PyMT animals. Presented data are the mean values ± SEM ( n = 11 mice per group). *** P < 0.001; Student’s t test. b Kaplan–Meier survival curves of mice treated with either vehicle ( n = 11 mice) or G28UCM agent ( n = 11 mice). Survival was calculated from the time of treatment initiation. Survival is increased in G28UCM-treated mice from 40 to 49 days (log-rank P < 0.01) . c Average animal weight during vehicle ( n = 11 mice) or treatment ( n = 11 mice). d Upper panels: FASN staining in vehicle- or G28UCM-treated breast tumors. Lower panels: collagen IV staining (basement marker) reveals that G28UCM-treated MMTV-PyMT animals develop only noninvasive ductal in situ carcinoma proliferations. Representative images were obtained for each treatment. Scale bar, 500 μm

Article Snippet: The following primary Abs were used: FASN (1:1000, clone: C20G5; 3180, Cell Signaling), PyMT (1:2500, NB100-2749, Novus Biological), K-Ras (1:1000, clone: F234; sc-30; Santa Cruz), HER2 (1:1000, 2242, Cell Signaling), IDH1 (1:1000, clone: D2H1, 8137, Cell Signaling), β-actin (1:5000, clone AC-15; A5441, Sigma), γ-tubulin (1:2500, clone GTU-88; T6557, Sigma), and vinculin (1:2000, clone: hVIN-1; V9131, Sigma).

Techniques: Inhibition, Staining, Marker, In Situ

Journal: bioRxiv

Article Title: Miro1 knockout inhibits mouse breast cancer tumorigenesis

doi: 10.1101/2025.05.06.650999

Figure Lengend Snippet: (A) Generation of the transgenic mouse model: Control mice have middle T-antigen activation and the experimental mouse model has concurrent Miro1 deletion and middle T-antigen activation. Cross breeding of the MIC mouse and the MTB mouse models with the Miro1 fl /fl murine model generated the final desired experimental mouse (MIC/ MTB/ Miro1 fl /fl). Activation of the mammary tumor virus long terminal repeat (MMTV-LTR) promoter in the presence of hormones results in reverse tetracycline trans activator (rtTA) production. In the presence of tetracycline (doxycycline) and rtTA, the tetracycline-inducible operator (TetO) is activated, leading to the simultaneous production of Polyomavirus middle T antigen (PyVMT) and CRE recombinase (CRE). An internal ribosome entry sequence (IRES) ensures simultaneous gene expression. (B) Presence of the transgenes were confirmed by genotyping an ear-clipping sample from each mouse used in the experiment. (C) IHC staining of Cre (where Cre is in brown and cell nuclei are in blue) confirmed expression in both WT and KO mice. IHC staining confirmed expression of Miro1 (where Miro1 is in brown and cell nuclei are in blue) in WT mice and lack of expression in KO mice. (D) IF staining of PyVMT (where PyVMT is yellow, and cell nuclei are pink) confirmed its expression in both the WT and KO mice. (E) percentage of tumor-free mice over time in days post-doxycycline administration. (F) Hematoxylin and eosin (H&E) staining of mammary epithelial tissue (where purple is cell nuclei and pink is cytoplasm) collected from wild type, fl /fl (Miro1 knockout), and heterozygous mice. (G) Scoring of tumors (1-5) removed from mice during necropsy using one representative tumor per mouse. Scoring of mammary epithelial tumor tissue based on our assessment of histological features such as loss of normal architecture, cellular morphology, and stromal invasion. .

Article Snippet: Sections from the same tissue samples were stained with an antibody for PyVMT (NOVUS #NB100-2749) to determine expression of the transgene.

Techniques: Transgenic Assay, Control, Activation Assay, Generated, Virus, Sequencing, Gene Expression, Immunohistochemistry, Expressing, Staining, Knock-Out

Journal: BMC Cancer

Article Title: Mechanisms for fibrate lipid-lowering drugs in enhancing bladder cancer immunotherapy by inhibiting CD276 expression

doi: 10.1186/s12885-025-14855-w

Figure Lengend Snippet: Fenofibrate (FNF) enhanced T cell-mediated antitumor immunity in bladder cancer. After T24 cells were pretreated with 0, 50, or 100 µM fenofibrate (FNF) for 24 h: A-B ELISA was used to detect the secretion levels of IFN-γ and TNF-α in the culture supernatants; C Pretreated T24 cells were co-cultured with activated T cells to evaluate the effect of FNF on T cell-mediated cytotoxicity against T24 cells; scale bar = 100 μm. ***, p < 0.001

Article Snippet: After incubation with 50 μM or 100 μM FNF for 24 h, cell culture supernatants were collected following centrifugation at 150 g for 15 min. IFN-γ (E-EL-H0108c) and TNF-α (E-EL-H0109c) concentrations in T24 cell supernatants were detected using ELISA kits purchased from Wuhan Elabscience Biotechnology Co., Ltd.

Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture