Journal: Molecular Metabolism

Article Title: Long-chain acyl-CoA synthetase-4 regulates endometrial decidualization through a fatty acid β-oxidation pathway rather than lipid droplet accumulation

doi: 10.1016/j.molmet.2024.101953

Figure Lengend Snippet: Inhibition of FA β-oxidation promotes lipid droplet accumulation. After MPA and db-cAMP-induced ESCs were treated with 10 μM, 20 μM FP-06424439 for 24 or 48 h, WB (A) was performed to detect the expression of CPT1A, CPT2, and ATP assay kit (B) was used to detect ATP level, ∗vs. NC. After MPA and db-cAMP-induced ESCs were transfected with DGAT2 siRNA, and WB (C) was performed to detect the expression of CPT1A, CPT2, and ATP assay kit (D) was used to detect ATP level, ∗vs. NC. E. The OCR was detected after MPA and db-cAMP-induced ESCs were treated with DGAT2 inhibitor PF-06424439 (20 μM) or/and DGAT1 inhibitor PF-04620110 (5 μM). F. Basal respiration, Basal respiration was calculated as the three OCR measurements before the addition of oligomycin minus the three OCR measurements after the addition of rotenone and antimycin A. G. ATP production, ATP production was calculated as the three OCR measurements before oligomycin injection minus the three OCR measurements after oligomycin injection. After MPA and db-cAMP-induced ESCs were treated with 25 μM etomoxir for 48 h, BIODIPY 493/503 staining (H, scale bar = 10 μm) was used to detect lipid droplet levels, and qPCR (I, normalized by GAPDH) was performed to detect the DGAT2 expression, ∗vs. NC, #vs. MPA + db-cAMP. After MPA and db-cAMP-induced ESCs were transfected with CPT1A siRNA, BIODIPY 493/503 staining (J, scale bar = 10 μm) was used to detect lipid droplet levels, and qPCR (K, normalized by GAPDH) was performed to detect the DGAT2 expression, ∗vs. NC, #vs. MPA + db-cAMP + NC siRNA. After MPA and db-cAMP-induced ESCs were transfected with OverACSL4 plasmid and treated with CPT1A siRNA or 25 μM etomoxir for 48 h, qPCR (L, normalized by GAPDH) or WB (M) was performed to detect the expression of PRL, IGFBP1, HOXA10, FOXO1, and FITC-phalloidin staining (N, scale bar = 50 μm) was performed to detect the F-actin expression, ∗vs. MPA + db-cAMP, #vs. MPA + db-cAMP + OverACSL4. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗ p < 0.001, # p < 0.05, ## p < 0.01, ### p < 0.001.

Article Snippet: Endometrial stromal cells induced to decidualization in vitro were treated with the carnitine palmitoyl transferase (CPT)1 inhibitor, etomoxir (25 μM, 50 μM; #HY-50202; MedChemExpress, Monmouth Junction, NJ, USA) for 24 h or 48 h; the diacylglycerol acyltransferase 2 (DGAT2) inhibitor, PF-06424439 (10 μM, 20 μM; #HY-108341A; MedChemExpress) for 24 h or 48 h; the DGAT1 inhibitor, PF-04620110 (5uM; #HY-13009; MedChemExpress) for 24 h; and CPT1 agonists, baicalin (25 μM, 50 μM; #HY-N0197; MedChemExpress), and C75 (1 μM, 4 μM; HY-12364, MedChemExpress) for 24 h, respectively.

Techniques: Inhibition, Expressing, ATP Assay, Transfection, Injection, Staining, Plasmid Preparation

Journal: Cell Reports

Article Title: Oxidative stress from DGAT1 oncoprotein inhibition in melanoma suppresses tumor growth when ROS defenses are also breached

doi: 10.1016/j.celrep.2022.110995

Figure Lengend Snippet: DGAT1 amplification and up-regulation is associated with poor prognosis in melanoma (A) Patient survival from TCGA melanoma cohort (25% top versus 75% bottom by mRNA abundance, y axis) versus fold change in mRNA expression of lipid metabolism genes in zebrafish tumors (x axis). (B) Kaplan-Meier survival plot comparing melanoma patients based on expression of DGAT1 or DGAT2 (top 25% versus bottom 75%, TCGA dataset). (C) DGAT1 and DGAT2 relative gene expression in skin, nevi, and melanoma tumors from indicated studies. Mean ± SD, n > 3. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. (D) Protein expression of DGAT1 and vinculin (loading control). NHM, normal human melanocytes. (E) Genetic alterations in the TCGA firehose legacy melanoma dataset (counting only samples with CNV data) obtained from cBioPortal. (F) Schematic depicting human chromosome 8, the amplified arm (red), and known/putative melanoma oncogenes within this region. (G) G-score of amplified regions of zebrafish chromosomes found in BRAF V600E -positive; tp53 mutant tumors indicating the position of presumed melanoma oncogene homologs. (H) Kaplan-Meier progression free-survival plot comparing patients across multiple cancer types based on DGAT1 amplification.

Article Snippet: AZD3988- DGAT1 Inhibitor , Tocris Bioscience , Cat. No. 4837.

Techniques: Amplification, Expressing, Gene Expression, Control, Mutagenesis

Journal: Cell Reports

Article Title: Oxidative stress from DGAT1 oncoprotein inhibition in melanoma suppresses tumor growth when ROS defenses are also breached

doi: 10.1016/j.celrep.2022.110995

Figure Lengend Snippet: DGAT1 functions as an oncoprotein in zebrafish and human melanocytes (A) DGAT1 amplification distribution in TCGA melanoma samples. (B) Kaplan-Meier plot of melanoma tumor nodule incidence in EGFP control or Dgat1a over-expressing animals on the tp53 mutant; nacre genetic background. Representative images shown for EGFP- and Dgat1a-positive animals at 54 and 76 weeks post-fertilization, respectively (mitfa:egfp n = 20; mitfa:dgat1a n = 20). (C) As for (B) but on the transgenic mitfa:BRAF V600E ; tp53 mutant; nacre genetic background. Representative images are shown at 12 weeks post-fertilization (mitfa:egfp n = 46; mitfa:dgat1a n = 21). (D) As for (C) but on the transgenic mitfa:NRAS G12D ; tp53 mutant; nacre genetic background, also shown the effect of Dgat2 over-expression (mitfa:egfp n = 120; mitfa:dgat1a n = 69; mitfa:dgat2 n = 35). (E) Hematoxylin and eosin-stained transverse sections of EGFP-expressing or Dgat1a-over-expressing melanoma on the transgenic mitfa : NRAS G12D ; tp53 mutant; nacre genetic background. Scale bars, 200 μm (F) Confluence of parental 888MEL cells and cells following lentiviral transduction with a DGAT1 over-expression vector and clonal selection (mean, n = 3, top left). Corresponding crystal violet staining after 72-h growth (top right). Corresponding protein expression of DGAT1 (bottom). (G) Relative cell numberof parental 888MEL cells and cells following lentiviral transduction with a DGAT1 over-expression vector, determined using crystal violet staining following 48-h culture in indicated serum levels under hypoxic (1% O 2 ) or normoxic conditions (mean ± SEM, n > 3). (H) Relative cell number of NHM and NHM following lentiviral transduction with a DGAT1 over-expression vector determined using crystal violet staining (left). Percentage of NHM and NHM following lentiviral transduction with a DGAT1 over-expression vector in S-phase by using EdU incorporation (right) (mean ± SEM, n = 5). (F–H) For significance: ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Article Snippet: AZD3988- DGAT1 Inhibitor , Tocris Bioscience , Cat. No. 4837.

Techniques: Amplification, Control, Expressing, Mutagenesis, Transgenic Assay, Over Expression, Staining, Transduction, Plasmid Preparation, Selection

Journal: Cell Reports

Article Title: Oxidative stress from DGAT1 oncoprotein inhibition in melanoma suppresses tumor growth when ROS defenses are also breached

doi: 10.1016/j.celrep.2022.110995

Figure Lengend Snippet: DGAT1 antagonism decreases melanoma cell proliferation and survival (A) Confluence of cell lines transfected with DGAT1 targeting (007, 008, pool) or scrambled (sc) siRNAs (mean, n = 3, top). Corresponding protein expression of DGAT1 (bottom). (B) Left: relative cell number (mean, n > 3) in indicated cell lines determined by crystal violet following 72-h DGAT1 inhibitor treatment (50 μM AZD3988, 30 μM A922500, 50 μM AZD7687, or 70 μM T863). Right: percentage of cells in S-phase by using EdU incorporation following 24-h DGAT1 inhibitor treatment (mean, n > 3). (C) Relative cell number determined by crystal violet staining following 72-h A922500 treatment with cells grown in varying concentrations of fetal calf serum (FCS; mean ± SD, n > 3). (D) Relative cell number determined by crystal violet staining following 48-h A922500 treatment under normoxic or hypoxic conditions (1% O 2 ) with cells grown in varying concentrations of FCS (relative to DMSO control for each condition; mean ± SD, n > 3). (E) Cleaved caspase index in indicated cell lines following transfection with either a DGAT1-targeting siRNA (007, 008, pool) or a sc control (mean, n = 3). (F) Protein expression of cleaved caspase-3 following treatment with/without A922500 for 24–72 h. (A) and (D) For significance: ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Article Snippet: AZD3988- DGAT1 Inhibitor , Tocris Bioscience , Cat. No. 4837.

Techniques: Transfection, Expressing, Staining, Control

Journal: Cell Reports

Article Title: Oxidative stress from DGAT1 oncoprotein inhibition in melanoma suppresses tumor growth when ROS defenses are also breached

doi: 10.1016/j.celrep.2022.110995

Figure Lengend Snippet: DGAT1-formed lipid droplets act as caretakers of mitochondrial health (A) Lipidomic profiling using UHPLC-MS of NRASG12D-positive EGFP-expressing (n = 6) and NRASG12D-positive Dgat1a-over-expressing (n = 6) tumors showing the ratio of individual lipid species annotated by MS/MS. (B) Representative images of 888MEL and Clone 3 cells stained with BODIPY (left; scale bar, 10 μm). Brightfield images of 888MEL parental cells and Clone 3 DGAT1-over-expressing cells (middle). UHPLC-lipidomic analysis of 888MEL parental and Clone 3 DGAT1-over-expressing cells. Fold change relative to 888MEL parental cells (right) (all conditions n = 3). TAG, triacylglycerides; AcCa, acylcarnitine. (C) Number of lipid droplets per cell visualized using BODIPY staining following AZD3988 (DGAT1i) treatment (mean ± SD, n > 30). (D) UHPLC-lipidomic analysis of SKMEL105 cells following A922500 treatment. Fold change relative to DMSO (all conditions n = 3). TAG, triacylglycerides; LPC, lysophosphatidycholine; LPE, lysophosphatidylethanolamine. (E) Lipid species fold changes in SKMEL105 following 72-h A922500 treatment plotted versus lipid species fold changes observed in Clone 3 cells. (F) Protein expression of phospho-AMPK and phospho-RAPTOR following A922500 (DGAT1i) treatment. (G) Oxygen consumption rate in A375 cells following 48-h A922500 treatment (top). Basal respiration, ATP production, and spare respiratory capacity were calculated (bottom) (mean ± SD, n = 3). (H) Staining with JC-1 dye following A922500 treatment or following transfection with DGAT1 targeting siRNA. The percentage of cells that lost red aggregates was calculated by using 1 μM CCP as a positive control and comparing this with untreated cells to create two populations of cells for flow cytometry analysis (top; mean ± SD, n > 3). Protein expression of PINK1 and PARKIN following A922500 treatment (bottom). (C, G, and H) For significance: ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Article Snippet: AZD3988- DGAT1 Inhibitor , Tocris Bioscience , Cat. No. 4837.

Techniques: Expressing, Tandem Mass Spectroscopy, Staining, Transfection, Positive Control, Flow Cytometry

Journal: Cell Reports

Article Title: Oxidative stress from DGAT1 oncoprotein inhibition in melanoma suppresses tumor growth when ROS defenses are also breached

doi: 10.1016/j.celrep.2022.110995

Figure Lengend Snippet: DGAT1 promotes survival of melanoma cells through suppressing ROS generation (A) Total proteomics workflow (left). GEO of up-regulated proteins (114) ranked by combined score (wikipathways) or log-adjusted p values (metascape, right). (DMSO n = 3, A922500 n = 3). (B) RT-qPCR analysis following A922500 treatment. Fold change relative to DMSO (mean, n = 3). (C) ROS levels quantified using dihydroethidium (DHE) fluorescence following A922500 (DGAT1i) treatment. Fold change relative to DMSO (n > 4). (D) C11-Bodipy staining following A922500 treatment. Mean fluorescence determined using FACS (mean ± SD, n > 6). (E) Mito-C11-Bodipy staining following A922500 treatment. Mean fluorescence determined using FACS (mean ± SD, n > 6). (F) 4-Hydroxynonenal (4HNE) protein conjugate abundance and protein expression of DGAT1 following transfection with either DGAT1-targeting siRNA or a scrambled control for 48 h. (G) 4HNE protein conjugate abundance and protein expression of DGAT1 following transfection with either DGAT1 over-expression vector or an empty vector control for 48 h. (H) 4HNE protein conjugate abundance and protein expression of Dgat1 and GFP in NRAS G12D -positive EGFP-expressing (n = 4) and NRAS G12D -positive Dgat1a-over-expressing (n = 7) tumors. (I) Cleaved-caspase index following transfection of a DGAT1-targeting or scrambled siRNA. At 24 h, cells were treated with/without Tempol or Ebselen (mean, n = 3, outer). Corresponding protein expression of DGAT1 and cleaved caspase-3 (inner). (C–E and I) For significance: ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Article Snippet: AZD3988- DGAT1 Inhibitor , Tocris Bioscience , Cat. No. 4837.

Techniques: Quantitative RT-PCR, Fluorescence, Staining, Expressing, Transfection, Control, Over Expression, Plasmid Preparation

Journal: Cell Reports

Article Title: Oxidative stress from DGAT1 oncoprotein inhibition in melanoma suppresses tumor growth when ROS defenses are also breached

doi: 10.1016/j.celrep.2022.110995

Figure Lengend Snippet: Combined DGAT1 and SOD1 inhibition halts tumor growth (A) Nude mice bearing A375 tumors were treated ( n = number of mice per group) with vehicle or A922500 (DGAT1i; 90 mg/kg/day) for 14 days. Data are presented as mean tumor volumes ±SD. (B) Quantification of ROS levels by DHE fluorescence in A375 cells following treatment with either A922500 or ATN-224 (SOD1i) alone or in combination (combo). Fold change relative to DMSO (mean ± SD, n = 5). (C) Relative number of A375 cells following treatment with either A922500 or ATN-224 alone or in combination. Fold change relative to DMSO (mean ± SD, n = 5). (D and E) Nude mice bearing A375 tumors (D) or MM485 tumors (E) were treated ( n = number of mice per group) with vehicle, A922500 (90 mg/kg/day) or TTM (SODi; 5 mg/kg/day) alone or in combination for 14 days. Data are presented as mean tumor volumes ±SD (top) or change in individual tumor volume (bottom). (F and G) 4HNE protein conjugate abundance and protein expression of SOD1 in A375 tumors (F) or MM485 tumors (G) treated with vehicle, A922500, or TTM alone or in combination. (B–E) For significance: ∗p < 0.05, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.

Article Snippet: AZD3988- DGAT1 Inhibitor , Tocris Bioscience , Cat. No. 4837.

Techniques: Inhibition, Fluorescence, Expressing

Journal: Cell Reports

Article Title: Oxidative stress from DGAT1 oncoprotein inhibition in melanoma suppresses tumor growth when ROS defenses are also breached

doi: 10.1016/j.celrep.2022.110995

Figure Lengend Snippet: Model Elevation of DGAT1 allows sequestration of fatty acids as triacylglycerides in lipid droplets and a controlled rate of fatty acid oxidation in mitochondria. If DGAT1 is suppressed, then fatty acids are oxidized, and cytotoxic reactive oxygen species are generated. However, these are neutralized through elevation of SOD1. However, if SOD1 is also suppressed, then the build-up of ROS is intolerable, and cells die.

Article Snippet: AZD3988- DGAT1 Inhibitor , Tocris Bioscience , Cat. No. 4837.

Techniques: Generated

Journal: Cell Reports

Article Title: Oxidative stress from DGAT1 oncoprotein inhibition in melanoma suppresses tumor growth when ROS defenses are also breached

doi: 10.1016/j.celrep.2022.110995

Figure Lengend Snippet:

Article Snippet: AZD3988- DGAT1 Inhibitor , Tocris Bioscience , Cat. No. 4837.

Techniques: Virus, Recombinant, Extraction, Multiplex sample analysis, Transfection, Staining, Imaging, cDNA Synthesis, RNA Extraction, Over Expression, Software

Journal: Nature Communications

Article Title: Cell cycle arrest induces lipid droplet formation and confers ferroptosis resistance

doi: 10.1038/s41467-023-44412-7

Figure Lengend Snippet: a Volcano plot displays log2 fold-change (FC) in lipid species abundance, comparing nocodazole-treated to vehicle-treated Caki-1 cells ( n = 510, P < 0.05, FC > 1.5). AcCa, acyl carnitine; CE, cholesterol ester; CER, ceramide; CL, cardiolipin; Co, coenzyme; DAG, diacylglyceride; HexCer, hexosyl ceramides; LPC, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; LPG, lysophosphatidylglycerol; LPI, lysophosphatidylinositol; PC, phosphatidylcholine; PC-O, ether-linked phosphatidylcholine; PE, phosphatidylethanolamine; PE-O/PE-P, ether-linked phosphatidylethanolamine; PA, phosphatidic acid; PG, phosphatidylglycerol; PI, phosphatidylinositol; PS, phosphatidylserine; SM, sphingomyelin; TAG, triacylglyceride; TAG-O, ether-linked triacylglyceride. b Boxplots displaying log2 FC in non-TAG ( n = 413 ) and TAG ( n = 97) lipid species abundance between nocodazole-treated and vehicle-treated Caki-1 cells, with medians and whiskers indicating min-max values. BODIPY 493/503 staining in Caki-1 cells after 48 h of cell cycle inhibitor treatment. scale bar, 20 μm ( c ), and quantifications in the indicated cell lines ( d ). BODIPY 493/503 staining in Caki-1 cells treated with iCDK4/6 for 48 h ( e ), or WT and sgCDK1 c e lls ( f ). g Triglyceride concentrations in Caki-1 cells. h Heat map of the significantly changed lipids (unpaired, two-tailed t -test; FDR-corrected P < 0.05, fold change > 1.5 in vehicle- versus nocodazole-treated Caki-1 cells). Rows represent samples, columns represent normalized intensities, color-coded from red (high intensity) to blue (low intensity). Volcano plots for iDGAT1/2-treated versus vehicle-treated ( i, n = 276) and iDGAT1/2 and nocodazole-treated versus nocodazole-treated (j, n = 302 ) Caki-1 cells. Log2 FC in phospholipid abundance comparing iDGAT and nocodazole-treated to nocodazole-treated Caki-1 cells, depicted with boxplots ( k ) and volcano plots ( l ). n = 192. LPC-O, ether-linked lysophosphatidylcholine; LPS, lysophosphatidylserine. BODIPY 493/503 staining in Caki-1 ( m ) and ACHN ( n ) cells. o Immunoblot of DGAT1 in Caki-1 cells. p BODIPY 493/503 staining in WT, DGAT DKO1, and DKO2 Caki-1 cells with or without exposure to hydroxyurea (0.3 mM) for 48 h. Mean ( ± SD) values are shown. n = 3. n indicates independent repeats, except a , b , i , j , k , and l . unpaired, two-tailed t -test. n.s. not significant. Source data are provided as a Source Data file.

Article Snippet: Cells were treated with cell cycle inhibitors, hydroxyurea (Sigma, #H8627), thymidine (Sigma, #T1895), colcemid (Sigma, #234109-M), nocodazole (Sigma, #487929-M), or the CDK4/6 inhibitor palbociclib (obtained from Dr. Keyomarsi); the DGAT1 inhibitor T863 (Sigma, #SML0539); the DGAT2 inhibitor PF06427878 (Sigma, #PZ0412); the ferroptosis inducers erastin (Cayman chemical, #17754), RSL3 (Tocris, #6118), or ML162 (Cayman chemical, #20455); the cell death inhibitors ferrostatin-1 (Sigma-Aldrich, #SML0583), necrostatin-1s (BioVision, #2263), or Z-VAD-fmk (R&D Systems, #FMK001); the antioxidant N -acetylcysteine (Sigma, #A9165); or the iron chelator deferoxamine (Sigma, #D9533).

Techniques: Staining, Two Tailed Test, Western Blot

Journal: Nature Communications

Article Title: Cell cycle arrest induces lipid droplet formation and confers ferroptosis resistance

doi: 10.1038/s41467-023-44412-7

Figure Lengend Snippet: Quantification of PI-positive Caki-1 cells treated with 2 μM erastin for 18 h using flow cytometry. Cells were pretreated with a vehicle or iDGAT1/2 with 0.3 mM hydroxyurea ( a ), 1 mM thymidine ( b ), 0.035 μg/ml colcemid ( c ), or 200 nM nocodazole ( d ) for 24 h. Lipid peroxidation measurement in Caki-1 cells treated with 2 μM erastin for 8 h. Cells were pretreated with a vehicle or iDGAT1/2 with 0.3 mM hydroxyurea ( e ), 1 mM thymidine ( f ), 0.035 μg/ml colcemid ( g ), or 200 nM nocodazole ( h ) for 24 h. WT, DGAT DKO1, and DGAT DKO2 Caki-1 cells were pretreated with cell cycle inhibitors for 24 h. Shown are the populations of PI-positive cells after treatment with 2 μM erastin for 18 h ( i – l ) and those with lipid peroxidation after treatment with 2 μM erastin for 8 h ( m – p ). q , r , The populations of PI-positive Caki-1 cells pretreated with a vehicle or iDGAT1/2 for 24 h after treatment with 2 μM erastin for 18 h ( q ) and lipid peroxidation after treatment with 2 μM erastin for 8 h ( r ). s , t WT, sgCDK1-2, and sgCDK1-3 Caki-1 cells were pretreated with iDGAT1/2 for 24 h. The populations of PI-positive cells after treatment with 2 μM erastin for 18 h ( s ) and lipid peroxidation after treatment with 2 μM erastin for 8 h ( t ) are shown. Mean ( ± SD) values are shown. n = 3. n indicates independent repeats (unpaired, two-tailed t -test). Source data are provided as a Source Data file.

Article Snippet: Cells were treated with cell cycle inhibitors, hydroxyurea (Sigma, #H8627), thymidine (Sigma, #T1895), colcemid (Sigma, #234109-M), nocodazole (Sigma, #487929-M), or the CDK4/6 inhibitor palbociclib (obtained from Dr. Keyomarsi); the DGAT1 inhibitor T863 (Sigma, #SML0539); the DGAT2 inhibitor PF06427878 (Sigma, #PZ0412); the ferroptosis inducers erastin (Cayman chemical, #17754), RSL3 (Tocris, #6118), or ML162 (Cayman chemical, #20455); the cell death inhibitors ferrostatin-1 (Sigma-Aldrich, #SML0583), necrostatin-1s (BioVision, #2263), or Z-VAD-fmk (R&D Systems, #FMK001); the antioxidant N -acetylcysteine (Sigma, #A9165); or the iron chelator deferoxamine (Sigma, #D9533).

Techniques: Flow Cytometry, Two Tailed Test

Journal: Redox Biology

Article Title: Chenodeoxycholic acid suppresses AML progression through promoting lipid peroxidation via ROS/p38 MAPK/DGAT1 pathway and inhibiting M2 macrophage polarization

doi: 10.1016/j.redox.2022.102452

Figure Lengend Snippet: CDCA causes LDs accumulation and lipid peroxidation through ROS/p38 MAPK/DGAT1 pathway in AML cells (A) Representative images of LDs in THP1 and Molm-13 detected by transmission electron microscope (TEM) were shown. The scale bar was 1 μm. (B) Representative images of LDs of THP1 (n = 3) and Molm-13 (n = 4) using BODIPY 493/503 measured by confocal microscope were shown. And the corresponding histograms were shown below the images. Scale bar = 10 μm. (C) Representative images of THP1 and Molm-13 using Oil Red O staining were shown. (D) Western blot analysis of DGAT1 protein after CDCA treatment was shown. (E) The representative images of LDs in THP1 and Molm-13 cells after 0, 0.12, 0.17 mmol/L CDCA treatment for 24h with or without A922500 pretreatment were shown (n = 3). And the corresponding histograms were shown on the right. (F) The representative images of LDs in THP1 and Molm-13 cells after being treated with 0, 0.12, 0.17 mmolm/L CDCA with or without NAC pretreatment were shown (n = 3). And the corresponding histograms were shown on the right. (G) Western blot analysis of DGAT1 in THP1 and Molm-13 cells after 0, 0.12, 0.17 mmol/L CDCA treatment for 24h with or without NAC pretreatment was shown. (H) The representative images of LDs in THP1 and Molm-13 cells after 0, 0.12, 0.17 mmol/L CDCA treatment for 24h with or without p38 inhibitor pretreatment were shown (n = 4). And the corresponding histograms were shown on the right. (I) Western blot analysis of DGAT1 in THP1 and Molm-13 cells after 0, 0.12, 0.17 mmol/L CDCA treatment for 24h with or without p38 inhibitor pretreatment was shown. (J) Representative images of THP1 (n = 4) and Molm-13 (n = 5) using BODIPY 581/591 C11(BD-C11) staining were shown. Red (591 nm) and green (488 nm) fluorescence indicate total and peroxidized lipids respectively. Quantitation of lipid peroxidation (the ratio of green to red) was exhibited on the right. Scale bar = 10 μm. (K) Quantitation of GSH levels are shown after CDCA treatment (n = 6). (L) Western blot analysis of GPX4 protein after CDCA treatment was shown. (M) The apoptosis analysis of the corresponding statistical histograms of THP1 (n = 3) and Molm-13 (n = 7) after ferroptosis inhibitor ferrostatin-1 treatment at concentrations of 0, 0.12, 0.17 mmol/L CDCA for 24h were shown. P-values were determined using student's t-test and Wilcoxon rank test. Error bars represent mean ± SEM. ns: Not significant; *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001. . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Besides, AML cells were pretreated with A922500 (MCE, China) for 1h before CDCA treatment to examine the level of LDs using flow cytometry.

Techniques: Transmission Assay, Microscopy, Staining, Western Blot, Fluorescence, Quantitation Assay

Journal: PLoS ONE

Article Title: Origin and Evolution of Retinoid Isomerization Machinery in Vertebrate Visual Cycle: Hint from Jawless Vertebrates

doi: 10.1371/journal.pone.0049975

Figure Lengend Snippet: A: Superposition of Lamprey LRATa and LRATb models on the H-REV107 crystal structure template (PyMol). Blue , Lamprey LRATa sequence. Green , Lamprey LRATb sequence. Magenta , 7-glycine motif on LRATb sequence. Side chains of a highly conserved LWNNCEHF motif with catalytic cysteine are shown in sticks, with red, oxygen; blue nitrogen and yellow , sulfur. B: Catalytic activity of Lamprey LRATb in HEK293F cells in the presence of specific DGAT1 inhibitor (A922500, 50 µM). Activity is expressed as ratio of retinyl esters to total retinoids (retinols and retinyl esters in %).

Article Snippet: DGAT1 specific inhibitor A922500 (Tocris) was added to cells to a final concentration 25–50 μM from a 10 mM stock in DMSO, together with all- trans retinol.

Techniques: Sequencing, Activity Assay

Journal: Diseases

Article Title: MOGAT2: A New Therapeutic Target for Metabolic Syndrome

doi: 10.3390/diseases3030176

Figure Lengend Snippet: MGAT2 inhibitors and structures.

Article Snippet: Currently, there are ongoing clinical studies using DGAT1 inhibitors (Novartis) to treat patients with familiar chylomicronemia [ ].

Techniques: In Vitro, Liquid Chromatography with Mass Spectroscopy

Journal: Cell reports

Article Title: Lipid droplet dynamics regulate adult muscle stem cell fate

doi: 10.1016/j.celrep.2021.110267

Figure Lengend Snippet: (A) UMAP embedding of scRNA-seq data on SCs isolated from non-injured muscles (n = 250) and injured muscles at 5 dpi (n = 2,835) and 10 dpi (n = 910), colored by isolation time. (B) UMAP embedding of scRNA-seq data colored by subpopulation identity. (C) Violin plot showing the expression of SC markers informing cluster annotation. (D and E) Heatmaps of the genes annotated in gene ontology (GO) term LD (GO: 0005811) and LD formation (GO: 0140042) (D), and neutral lipid (GO: 0046460) and TAG biosynthetic process (GO: 0019432) and (E) in SC clusters. (F) Density plot visualizing enrichment of genes in GO term LD and neutral lipid biosynthetic process in SC clusters. (G) Violin plot showing the expression of key LD biosynthesis-related genes. (H and I) Inhibition of LD biogenesis in SCs with DGAT inhibitors (DGATin) (H) and quantification of PAX7 + SCs per cluster (I) on cultured single myofibers at 72 h (n = 5 mice). Scale bar, 10 μm. (J and K) Immunostaining PAX7 and MyoD (J) and quantification of SC population on 72 h cultured single myofibers (K) (n = 5 mice). Scale bar, 10 μm. (L and M) Immunostaining of MyoG (L) and quantification of MyoG + cells per myofiber (M) with control and DGATin treatment for 72 h (n = 3 mice). Scale bar, 20 μm. Data are mean ± standard deviation; Student’s t test. **p < 0.01, ***p < 0.001. See also .

Article Snippet: For the inhibition of LD biogenesis, single myofibers were cultured with 1 μM DGAT1 inhibitor (A922500, Sigma-Aldrich, cat#A1737,) and 2.5 μM DGAT2 inhibitor (PF-06424439, Sigma-Aldrich, cat#PZ0233) for 72 h, immediately after isolation.

Techniques: Isolation, Expressing, Inhibition, Cell Culture, Immunostaining, Standard Deviation

Journal: Cell reports

Article Title: Lipid droplet dynamics regulate adult muscle stem cell fate

doi: 10.1016/j.celrep.2021.110267

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: For the inhibition of LD biogenesis, single myofibers were cultured with 1 μM DGAT1 inhibitor (A922500, Sigma-Aldrich, cat#A1737,) and 2.5 μM DGAT2 inhibitor (PF-06424439, Sigma-Aldrich, cat#PZ0233) for 72 h, immediately after isolation.

Techniques: Recombinant, Lysis, Modification, Staining, Blocking Assay, Plasmid Preparation, Transfection, Isolation, Bicinchoninic Acid Protein Assay, Western Blot, Detection Assay, Multiplex Assay, Software, Microscopy