fasn inhibitor  (MedChemExpress)

Journal: bioRxiv

Article Title: An alternative route for β-hydroxybutyrate metabolism supports fatty acid synthesis in cancer cells

doi: 10.1101/2024.10.31.621317

Figure Lengend Snippet: A , Schematic of β-OHB metabolism and 13 C labeling derived from [U- 13 C]-β-OHB. AcAc, acetoacetate; BDH1, β-OHB dehydrogenase 1; OXCT1, 3-oxoacid CoA-transferase 1; OAA, oxaloacetate. B , Immunoblot for BDH1, OXCT1, and vinculin in the indicated cancer cell lines. C , Proliferation rates of the indicated cancer cell lines grown in high or low glucose conditions, with or without 5 mM β-OHB. D , Proliferation rates of the indicated cancer cell lines grown in lipid-replete versus lipid-depleted culture media, with or without 5 mM β-OHB. E , Proliferation rates of the indicated cancer cell lines grown in lipid-depleted media, with or without 5 mM β-OHB and 0.3 µM of the FASN inhibitor GSK2194069. Data are presented as mean ± s.e.m; n = 3 biologically independent replicates. Comparisons were made using a two-tailed Student’s t test ( C-E ). * P<0.05 , ** P<0.01 , *** P<0.001 .

Article Snippet: The fatty acid synthase (FASN) inhibitor GSK2194069 (Tocris, 5303) was used at the indicated concentrations.

Techniques: Labeling, Derivative Assay, Western Blot, Two Tailed Test

Journal: Cell reports

Article Title: Hepatocyte vitamin D receptor functions as a nutrient sensor that regulates energy storage and tissue growth in zebrafish

doi: 10.1016/j.celrep.2024.114393

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: GSK2194069 , Cayman Chemical Company , Cat#20022.

Techniques: Recombinant, Concentration Assay, TA Cloning, Sequencing, Cloning, DNA Library Preparation, Reverse Transcription, SYBR Green Assay, Quantitation Assay, CRISPR, Plasmid Preparation, Software

Journal: Cell reports

Article Title: Hepatocyte vitamin D receptor functions as a nutrient sensor that regulates energy storage and tissue growth in zebrafish

doi: 10.1016/j.celrep.2024.114393

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: GSK2194069 (Cayman Chemical Company) stock solution was prepared in DMSO at 10 mM GSK2194069 exposure (10 μM) was conducted from 5 to 7 dpf.

Techniques: Recombinant, Concentration Assay, TA Cloning, Sequencing, Cloning, DNA Library Preparation, Reverse Transcription, SYBR Green Assay, Quantitation Assay, CRISPR, Plasmid Preparation, Software

Journal: Nature Communications

Article Title: A fast-acting lipid checkpoint in G1 prevents mitotic defects

doi: 10.1038/s41467-024-46696-9

Figure Lengend Snippet: A Scatter plots show example correlations between relative lipid abundance and S phase percentage over the different inhibitor treatments and time points. Line indicates linear fit. Dots are labeled based on inhibitor treatment and time point. B Left: Nodes of the network are color-coded based on the correlations between relative lipid abundance and S phase percentage. Right: For orientation the lipid map shows the nodes of the network color-coded by lipid classes. A , B Data are combined of at least three independent experiments and shown as mean. C Top: Hierarchical clustering of lipid-mRNA abundance correlations. Rows: 250 lipids, columns: 3739 differentially expressed genes after C75 treatment. Bottom: Rug plot represents the distribution of ATF4-dependent genes and the gray area shows the percentage of ATF4-dependent genes per gene cluster. 3739 genes are divided into 11 gene clusters (clustergram - MATLAB) based on their lipid correlation. Clusters 5 and 6 are significantly enriched for ATF-dependent genes as calculated by two-sided Fisher’s exact test. D – F Example correlations of selected genes with 250 lipids color-coded on the circular network. Different clusters show different distribution of correlations across the network. G Bar plot shows fold enrichment for Gene Ontology (GO) Terms (BP direct) among genes significantly upregulated in cells treated with C75 for 3 h at 9 h after EGF release (mRNA sequencing data, significant in at least 3 replicates, p.adj <0.05, filtered for increased expression in starved cells and at least twofold upregulated compared to DMSO control). P values were calculated using EASE score (Fisher’s Exact test). Processes sorted by false discovery rate (FDR)–adjusted P value. Redundant processes omitted. H Volcano plot shows differentially regulated genes after 3 h of C75 treatment. Differentially expressed genes (log 2 (fold-change) | > 1, p.adj. <0.014) in blue (genes associated with the ER stress pathway based on GO terms) or magenta (ATF4-dependent genes ) or both (blue-magenta). CDKN1A (p21) is highlighted in red as the ER stress-cell cycle link. G , H P. adj. values are Benjamini-Hochberg adjusted p values calculated using the two-sided Wald test. Inhibitor concentrations used: C75 (30 µM), GSK2194069 (50 µM), SCDi (32 µM). FC, fold-change.

Article Snippet: Chemicals used were C75 (Sigma Aldrich), TOFA (5-(tetradecyloxy)-2-furoic acid), acetyl-CoA carboxylase inhibitor (Abcam, ab141578), Triascin C (Enzo, BML-EI218-100), Bromoenol Lactone (Cayman Chemical, 70700), Etomoxir (Calbiochem/MilliporeSigma), T-863 (Cayman Chemical, 25807), SB 204990 (Cayman Chemical, 15245), Cerulenin (Santa Cruz Biotechnology), GSK2194069 (Sigma Aldrich), CAY10566, GSK2606414 (both Cayman Chemical), Tunicamycin (Cayman Chemical), MMS (Santa Cruz Biotechnology), NCS (Sigma Aldrich), Torin2, Nutlin-3 (both Medchemexpress), DMSO (Sigma Aldrich).

Techniques: Labeling, Sequencing, Expressing, Control

Journal: Nature Communications

Article Title: A fast-acting lipid checkpoint in G1 prevents mitotic defects

doi: 10.1038/s41467-024-46696-9

Figure Lengend Snippet: A EGF-released MCF-10A cells (4 h) treated with DMSO or C75. Sample immunofluorescence images for nuclei (Hoechst), p21, and Cyclin D are shown. Scale bar: 10 µm. Data are representative of at least seven independent experiments. B Quantification of nuclear protein levels as shown in ( A ). Data are shown as mean ± SD from at least seven independent experiments, n > 20,000 cells per condition. C EGF-released cells (4 h) treated with indicated inhibitors. Blotted for Cyclin D, p21, and β-actin (loading control). Data are representative of three independent experiments. D Nuclear p21 and Cyclin D protein levels after EGF release and treatment with DMSO or C75 measured by immunofluorescence. Data are representative of at least two independent experiments (data points are means of two technical replicates), n > 17,000 cells per condition. E mRNA levels of CDKN1A (p21) and CCND1 (Cyclin D) measured by qRT-PCR after treatment with DMSO or C75 and normalized to unreleased cells. Data are from at least two independent experiments. F Cyclin D and p21 nuclear protein levels measured by immunofluorescence after 8 h of EGF release in the presence of DMSO or Tunicamycin. Data are from two independent experiments, n > 12,000 cells per condition. B , E, F P values calculated using two-tailed Paired t test. Inhibitor concentrations used unless indicated otherwise: C75 (15 µM), GSK2194069 (50 µM), Tunicamycin (10 µg/ml), Nutlin-3 (10 µM), Torin2 (500 nM). Source data are provided as Source data file.

Article Snippet: Chemicals used were C75 (Sigma Aldrich), TOFA (5-(tetradecyloxy)-2-furoic acid), acetyl-CoA carboxylase inhibitor (Abcam, ab141578), Triascin C (Enzo, BML-EI218-100), Bromoenol Lactone (Cayman Chemical, 70700), Etomoxir (Calbiochem/MilliporeSigma), T-863 (Cayman Chemical, 25807), SB 204990 (Cayman Chemical, 15245), Cerulenin (Santa Cruz Biotechnology), GSK2194069 (Sigma Aldrich), CAY10566, GSK2606414 (both Cayman Chemical), Tunicamycin (Cayman Chemical), MMS (Santa Cruz Biotechnology), NCS (Sigma Aldrich), Torin2, Nutlin-3 (both Medchemexpress), DMSO (Sigma Aldrich).

Techniques: Immunofluorescence, Control, Quantitative RT-PCR, Two Tailed Test

Journal: Nature Communications

Article Title: A fast-acting lipid checkpoint in G1 prevents mitotic defects

doi: 10.1038/s41467-024-46696-9

Figure Lengend Snippet: A Schematic highlighting Cyclin-dependent kinase (CDK) signaling in G1. B Histogram of Rb(p-S807/S811) signal of cells treated with C75 or DMSO after EGF release (20 h). Percentages are shown as mean ± SD of four independent experiments, n > 17,000 cells per condition. C Dose response of percent Rb(p-S807/S811) positive cells treated with C75 normalized to DMSO treatment after EGF release (20 h). Data are from at least three independent experiments, n > 17,000 cells per condition. D Percentage of Rb(p-S807/S811) positive cells treated with DMSO or C75 after EGF release (20 h). GM (growth media): 5% serum. Data are shown as mean ± SD from three independent experiments, n > 15,000 cells per condition. E Percentage of Rb(p-S807/S811) positive cells treated with DMSO or C75 after EGF release (20 h). PA-BSA indicates the presence of Palmitate complexed to BSA. NaCl-BSA is the control treatment. Data are shown as mean ± SD from three independent experiments, n > 15,000 cells per condition. F Histogram of Rb(p-S807/S811) signal of cells treated with Tunicamycin or DMSO and EGF-released (20 h). Percentages are shown as mean ± SD of at least three independent experiments, n > 15,000 cells per condition. G Percent of Rb(p-S807/S811) positive cells transfected with sicontrol (sictrl) or sip21 and treated with DMSO or C75. Data are shown as mean ± SD from three independent experiments, n > 14,000 cells per condition. H Schematic depicting the workflow of treatments. Histogram of Rb(p-S807/S811) signal of cells treated with different inhibitors. Percentages are from two independent experiments and shown as mean ± SD, n > 20,000 cells per condition. I Schematic summary of the findings: Lipidome analysis shows the balance of increased unsaturated lipids (blue) and decreased saturated lipids (red) to overcome the lipid checkpoint in G1 (outlook for S/G2/M is extrapolated based on the G1 data). Lipid checkpoint engagement mediated by PERK-mediated ER stress feeds back to cell-cycle signaling by increasing p21 levels, decreasing Cyclin D and Rb (p-S807/S811) causing a cell-cycle delay. D , E , G P values calculated using two-tailed Paired t test. Inhibitor concentrations used unless indicated otherwise: C75 (15 µM), GSK2194069 (50 µM), Tunicamycin (10 µg/ml), Palmitate (5 µM). Source data are provided as Source data file.

Article Snippet: Chemicals used were C75 (Sigma Aldrich), TOFA (5-(tetradecyloxy)-2-furoic acid), acetyl-CoA carboxylase inhibitor (Abcam, ab141578), Triascin C (Enzo, BML-EI218-100), Bromoenol Lactone (Cayman Chemical, 70700), Etomoxir (Calbiochem/MilliporeSigma), T-863 (Cayman Chemical, 25807), SB 204990 (Cayman Chemical, 15245), Cerulenin (Santa Cruz Biotechnology), GSK2194069 (Sigma Aldrich), CAY10566, GSK2606414 (both Cayman Chemical), Tunicamycin (Cayman Chemical), MMS (Santa Cruz Biotechnology), NCS (Sigma Aldrich), Torin2, Nutlin-3 (both Medchemexpress), DMSO (Sigma Aldrich).

Techniques: Control, Transfection, Two Tailed Test

Journal: Cancers

Article Title: Co-Targeting FASN and mTOR Suppresses Uveal Melanoma Growth.

doi: 10.3390/cancers15133451

Figure Lengend Snippet: Figure 3. Co-targeting FASN and mTOR reduces UM cell growth. (A) 92.1, UM001 and UM004 cells were treated with Fasnall (0, 2.5, 5 and 7.5 µM) for 4 days or GSK 2194069 (0, 20, 40 and 60 µM) for 3 days. Cell viability was measured by crystal violet staining. Quantification of cell growth following treatment with Fasnall and GSK2194069 is shown as fold changes in crystal violet stain compared to controls. (B) FASN knockdown was performed by siRNA transfection. Silencing of FASN was confirmed through Western blot. (C) Activation of mTOR modulates de novo lipogenesis in cancer cells by controlling the expression of transcription factor (SREBP1) and key lipogenic enzymes (e.g., ACLY, ACC and FASN; colored red). (D) Phosphorylated and total mTOR levels in NCMs and UM cell lines were probed by Western blot. β-actin served as a loading control. (E) The effects of co-inhibition of FASN and mTOR in 92.1, UM001 and UM004 cell growth were measured by IncuCyte. The cells were treated with Fasnall (5 µM) or GSK2194069 (40 µM), with or without AZD2014 (200 nM), for 72 h. Percent confluency of the cells was measured on day 0 and day 3. Data are shown as mean ± SEM (n = 4) * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 unpaired t-test. NCMs; normal choroidal melanocytes, mTOR; mammalian target of rapamycin, ACLY; ATP- citrate lyase, ACC; acetyl-CoA carboxylase, FASN; fatty acid synthase and SREBP1; sterol regulatory element-binding protein.

Article Snippet: Fasnall (Sigma-Aldrich), GSK2194069, Fatostatin (Tocris, Minneapolis, MN, USA) and AZD2014 (Selleck Chemicals, Houston, TX, USA) were purchased, and the inhibitors were dissolved in DMSO.

Techniques: Staining, Knockdown, Transfection, Western Blot, Activation Assay, Expressing, Control, Inhibition, Binding Assay

Journal: Cancers

Article Title: Co-Targeting FASN and mTOR Suppresses Uveal Melanoma Growth.

doi: 10.3390/cancers15133451

Figure Lengend Snippet: Figure 4. Effects of FASN and mTOR inhibitors on levels of cell cycle modulators in UM. UM004, 92.1 and UM001 cells were treated with Fasnall (5 µM) or GSK2194069 (40 µM), with or without AZD2014 (200 nM), for 48 h. (A) RPPA data were used to determine proteins/phospho-proteins that were significantly different between control, single treatments of each inhibitor and combo treatments of different cell lines (p-value < 0.05 and a 25% log2 fold change). Comparisons were performed between each group using the two-sample t-test method with 1000 permutations and assumed unequal variance. Hierarchical clustering was performed based on median-centered log2- transformed expression values. Statistical calculations were performed in Matlab® (v2015b) using the mattest function. (B) Results of RPPA analysis were validated by Western blot in UM001 and UM004 cells. β-actin, HSP90 and S6 served as loading controls. Protein expression was normalized to the average intensity of the loading controls.

Article Snippet: Fasnall (Sigma-Aldrich), GSK2194069, Fatostatin (Tocris, Minneapolis, MN, USA) and AZD2014 (Selleck Chemicals, Houston, TX, USA) were purchased, and the inhibitors were dissolved in DMSO.

Techniques: Control, Transformation Assay, Expressing, Western Blot

Journal: Cancers

Article Title: Co-Targeting FASN and mTOR Suppresses Uveal Melanoma Growth.

doi: 10.3390/cancers15133451

Figure Lengend Snippet: Figure 5. Inhibition of FASN and mTOR induce cell cycle arrest in 2D and 3D cell growth. UM004, 92.1 and UM001 cells were treated with Fasnall (5 µM) or GSK2194069 (40 µM), with or without AZD2014 (200 nM), for 48 h. (A) Cells were collected for EdU corporation (upper panel) and annexin/PI staining assays (bottom panel). (B) 3D spheroid cultures of 92.1 cells were treated with Fasnall (5 µM) or GSK2194069 (40 µM), with or without AZD2014 (200 nM), for 48 h. EdU incorporation of 92.1 grown as 3D spheroids is shown. (C) Representative figures of spheroids are shown. Tumor spheroids were treated with Fasnall (5 µM) or GSK2194069 (40 µM), with or without AZD2014 (200 nM), for 48 h. For cell viability, 3D spheroids were stained with Calcein-AM and PI for live cells and necrotic cells, respectively. (D) Quantitation of Calcein-AM and PI. Magnification: 150X, Scale bar: 100 µm. Quantification bar graph is shown. Data are shown as mean ± SEM (n = 4). * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 unpaired t-test.

Article Snippet: Fasnall (Sigma-Aldrich), GSK2194069, Fatostatin (Tocris, Minneapolis, MN, USA) and AZD2014 (Selleck Chemicals, Houston, TX, USA) were purchased, and the inhibitors were dissolved in DMSO.

Techniques: Inhibition, Staining, Quantitation Assay

Journal: Cancers

Article Title: Co-Targeting FASN and mTOR Suppresses Uveal Melanoma Growth.

doi: 10.3390/cancers15133451

Figure Lengend Snippet: Figure 6. Suppression of FASN and mTOR decrease de novo FA biosynthesis in UM cells. UM001 and OMM1.3 cells were incubated in the presence of 13C-glucose for 4 and 24 h along with GSK2194069 (40 µM) and with or without AZD2014 (200 nM). (A) A flow of carbon atoms from the glucose to the fatty acid palmitate, including depictions of M + 4 through M + 16 isotopologues. Black circles = carbon-13; white circles = carbon-12. (B) Fractional labeling of myristate (14:0), palmitate (16:0), palmitoleate (16:1) and stearate (18:0) in UM cells at 24 h after the addition of 13C-glucose. Data are shown as mean ± SD (n = 3). ns, not significant, ** p < 0.01, and **** p < 0.0001 Two-way ANOVA with Dunnett’s multiple comparison testing. (C) Isotopologue distribution patterns of palmitate from 13C-glucose in UM001 and OMM1.3 cells at 4 h (left panel) and 24 h (right panel). Data are shown as mean ± SD (n = 3).

Article Snippet: Fasnall (Sigma-Aldrich), GSK2194069, Fatostatin (Tocris, Minneapolis, MN, USA) and AZD2014 (Selleck Chemicals, Houston, TX, USA) were purchased, and the inhibitors were dissolved in DMSO.

Techniques: Incubation, Labeling, Comparison

Journal: Cancers

Article Title: Co-Targeting FASN and mTOR Suppresses Uveal Melanoma Growth.

doi: 10.3390/cancers15133451

Figure Lengend Snippet: Figure 7. The effects of FASN and mTOR inhibitors on glucose utilization in UM cells. UM001 and OMM1.3 cells were incubated in the presence of 13C-glucose for 4 and 24 h along with GSK2194069 (40 µM) and with or without AZD2014 (200 nM). (A) Net flux of glucose consumption and lactate secretion from UM cells calculated at fmol/hour/cell. Data are shown as mean ± SD (n = 3). ns, not significant, * p < 0.05, ** p < 0.01, and *** p < 0.001. Two-way ANOVA with Dunnett’s multiple comparison testing. (B) Glycolytic capacity of UM001 and OMM1.3 cells after the treatment (24 h) was measured by ECAR using the Seahorse analyzer. Data were normalized to protein level and analyzed via Agilent Seahorse XF report generators. Data are shown as mean ± SEM (n = 12). ns, not significant, * p < 0.05, and ** p < 0.01.

Article Snippet: Fasnall (Sigma-Aldrich), GSK2194069, Fatostatin (Tocris, Minneapolis, MN, USA) and AZD2014 (Selleck Chemicals, Houston, TX, USA) were purchased, and the inhibitors were dissolved in DMSO.

Techniques: Incubation, Comparison

Journal: Cancers

Article Title: Co-Targeting FASN and mTOR Suppresses Uveal Melanoma Growth.

doi: 10.3390/cancers15133451

Figure Lengend Snippet: Figure 8. Inhibition of FASN and mTOR decrease the flux of glucose into the TCA cycle in UM cells. UM001 and OMM1.3 cells were incubated in the presence of 13C-glucose for 4 and 24 h along with GSK2194069 (40 µM) and with or without AZD2014 (200 nM). Fractional labeling of TCA cycle intermediates in UM001 and OMM1.3 cells (4 and 24 h) after the addition of 13C-glucose. Inset numbers indicate percent labeling for each isotopologue (n = 3).

Article Snippet: Fasnall (Sigma-Aldrich), GSK2194069, Fatostatin (Tocris, Minneapolis, MN, USA) and AZD2014 (Selleck Chemicals, Houston, TX, USA) were purchased, and the inhibitors were dissolved in DMSO.

Techniques: Inhibition, Incubation, Labeling