Journal: Molecular cell

Article Title: Probing the Global Cellular Responses to Lipotoxicity Caused by Saturated Fatty Acids

doi: 10.1016/j.molcel.2019.01.036

Figure Lengend Snippet: (A) Schematic representation of the SREBP pathway in which GP78 degrades INSIG1 to free the SCAP-SREBP1 complex for trafficking from the ER to the Golgi apparatus, where cleavage liberates the SREBP1 transcription factor fragment from the membrane. This, in turn, leads to induction of target genes that have a sterol-response element (SRE) sequence in their promoter, such as SCD1. Aggravating hits are shown in red, protective hits are shown in blue. (B) INSIG1 knockdown elevates FAS and SCD1 and reduces palmitate-induced upregulation of BIP. Western blot of WT and INSIG1-kd cells under basal condition or after palmitate treatment (0.15 mM, 20 h). (C) INSIG1 knockdown modestly protects cells from palmitate-induced cell death, as measured by PI staining, and this protection is lost with SCD inhibition (4 μM). **p < 0.001. (D) Unfolded protein response gene expression levels of WT and INSIG1-kd cells. (E) Relative quantification for phosphatidic acid (PA, left panel) and diacylglycerol (DAG, right panel) in INISIG1-kd cells compared to WT cells before after after palmitate treatment, as identified by LC-MS2. K562 cells untreated or treated with 0.2 mM palmitate. n=3–4 for each treatment. ***p < 0.001. (F) INSIG1-kd cells exhibit increased incorporation of radiolabeled palmitate into TG. Cells were treated for 6 h with 0.2 mM nonradiolabeled palmitate and 0.15μCi 14C-palmitate. Triacsin C (10uM, an inhibitor of ACSL1, ACSL3 and ACSL4) was added confirm ACSL-specific fatty acid uptake. Untreated and triacsin C values are also shown in Figure 1H. n=3 for each treatment. **p < 0.01; ***p < 0.001. See also Figures S4.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies XBP1 Biolegend Cat # 619501 Caspase-3 Cell Signaling Cat # 966 Caspase-8 Cell Signaling Cat # 9746 SAPK/JNK Cell Signaling Cat # 9252 Phosphor-SAPK/JNK (Thr183/185) (G9) Cell Signaling Cat # 9255 Actin Cell Signaling Cat # 3700 Tubulin Cell Signaling Cat # 5346 SCD1 Cell Signaling Cat # 2438 Cytochrome b5 Cell Signaling Cat # 30311 Cytochrome b5 reductase Sigma SAB4500655 Vinculin Cell Signaling Cat # 4650 BiP Cell Signaling Cat # 3177 Calnexin Cell Signaling Cat # 2679 FAS Cell Signaling Cat # 3180 RNF213 Sigma HPA026790 INSIG1 This paper N/A Chemicals, Peptides, and Recombinant Proteins BODIPY (493/503) Thermo Fisher Cat # D3922 SCD inhibitor Abcam Cat #142089 DGAT1 inhibitor Merck & Co., Inc. Liu et al., 2013 Triacsin C Santa Cruz Cat # sc-200574A 4u8c EMD Millipore Cat # 412512 GSK2606414 Tocris Cat # 5107 iScript cDNA Synthesis Kit BioRad Cat # 170-8891 Power SYBR Green PCR Master Mix Life Technologies Cat # 4368706 RNeasy RNA isolation kit Qiagen Cat # 79306 1,2-dioleoyl-sn-glycerol Avanti 800811O 1,2-dipalmitoyl-sn-glycerol Avanti 800816 Stearoyl [9,10-3H] Coenzyme A American Radiolabeled Chemicals, Inc. ART 0390 Palmitic Acid [1-14C] American Radiolabeled Chemicals, Inc. ARC 0172A Stearoyl coenzyme A Sigma Aldrich Cat # S0802 NADH Sigma Aldrich Cat # N8129 Protease inhibitor cocktail Sigma-Aldrich 118736170001 PhosSTOP phosphatase inhibitor Sigma-Aldrich 4906837001 Critical Commercial Assays Dead Cell Apoptosis Kit Thermo Fisher V13242 Experimental Models: Cell Lines K562 ATCC CCL-243 SUM159 Laboratory of Tomas Kirchhausen RRID:CVCL_5423 HeLa Laboratory of Wade Harper N/A HepG2 ATCC N/A Oligonucleotides RNF213 siRNA Dharmacon D-001220-01-05 RISC-free (Control) siRNA Dharmacon L-023324-00-0005 For primers for qPCR and cloning, see Table S5 This paper N/A Deposited Data Raw and processed RNAseq data NCBI GEO GSE125178 Software and Algorithms Lipid Search Thermo Fisher Taguchi et al., 2010 ClueGo Cytoscape http://apps.cytoscape.org/apps/cluego Open in a separate window KEY RESOURCES TABLE.

Techniques: Membrane, Sequencing, Knockdown, Western Blot, Staining, Inhibition, Gene Expression, Quantitative Proteomics

Journal: Molecular cell

Article Title: Probing the Global Cellular Responses to Lipotoxicity Caused by Saturated Fatty Acids

doi: 10.1016/j.molcel.2019.01.036

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies XBP1 Biolegend Cat # 619501 Caspase-3 Cell Signaling Cat # 966 Caspase-8 Cell Signaling Cat # 9746 SAPK/JNK Cell Signaling Cat # 9252 Phosphor-SAPK/JNK (Thr183/185) (G9) Cell Signaling Cat # 9255 Actin Cell Signaling Cat # 3700 Tubulin Cell Signaling Cat # 5346 SCD1 Cell Signaling Cat # 2438 Cytochrome b5 Cell Signaling Cat # 30311 Cytochrome b5 reductase Sigma SAB4500655 Vinculin Cell Signaling Cat # 4650 BiP Cell Signaling Cat # 3177 Calnexin Cell Signaling Cat # 2679 FAS Cell Signaling Cat # 3180 RNF213 Sigma HPA026790 INSIG1 This paper N/A Chemicals, Peptides, and Recombinant Proteins BODIPY (493/503) Thermo Fisher Cat # D3922 SCD inhibitor Abcam Cat #142089 DGAT1 inhibitor Merck & Co., Inc. Liu et al., 2013 Triacsin C Santa Cruz Cat # sc-200574A 4u8c EMD Millipore Cat # 412512 GSK2606414 Tocris Cat # 5107 iScript cDNA Synthesis Kit BioRad Cat # 170-8891 Power SYBR Green PCR Master Mix Life Technologies Cat # 4368706 RNeasy RNA isolation kit Qiagen Cat # 79306 1,2-dioleoyl-sn-glycerol Avanti 800811O 1,2-dipalmitoyl-sn-glycerol Avanti 800816 Stearoyl [9,10-3H] Coenzyme A American Radiolabeled Chemicals, Inc. ART 0390 Palmitic Acid [1-14C] American Radiolabeled Chemicals, Inc. ARC 0172A Stearoyl coenzyme A Sigma Aldrich Cat # S0802 NADH Sigma Aldrich Cat # N8129 Protease inhibitor cocktail Sigma-Aldrich 118736170001 PhosSTOP phosphatase inhibitor Sigma-Aldrich 4906837001 Critical Commercial Assays Dead Cell Apoptosis Kit Thermo Fisher V13242 Experimental Models: Cell Lines K562 ATCC CCL-243 SUM159 Laboratory of Tomas Kirchhausen RRID:CVCL_5423 HeLa Laboratory of Wade Harper N/A HepG2 ATCC N/A Oligonucleotides RNF213 siRNA Dharmacon D-001220-01-05 RISC-free (Control) siRNA Dharmacon L-023324-00-0005 For primers for qPCR and cloning, see Table S5 This paper N/A Deposited Data Raw and processed RNAseq data NCBI GEO GSE125178 Software and Algorithms Lipid Search Thermo Fisher Taguchi et al., 2010 ClueGo Cytoscape http://apps.cytoscape.org/apps/cluego Open in a separate window KEY RESOURCES TABLE.

Techniques: Recombinant, cDNA Synthesis, SYBR Green Assay, Isolation, Protease Inhibitor, Control, Cloning, Software

Journal: Journal of Lipid Research

Article Title: Identification and characterization of LPLAT7 as an sn -1-specific lysophospholipid acyltransferase

doi: 10.1016/j.jlr.2022.100271

Figure Lengend Snippet: sn -1 LPLAT activities are detected in a wide range of cells and tissues. A–C, sn -1 LPLAT activities in various mouse tissues and cells. A: LPLAT assays were performed using sn -2-rich LPC (oleoyl (C18:1) LPC ( Fig. 1 A)) as acyl acceptors, stearoyl-CoA (C18:0-CoA, upper), and palmitoyl-CoA (C16:0-CoA, lower) as acyl donors and membrane fractions of various mouse tissues as enzyme sources. B, C, LPLAT assays were performed using sn-2 -rich LPC (C18:1 LPC) and sn -1-rich LPC (stearoyl (C18:0) LPC) as acyl acceptors, C18:0-CoA, C16:0-CoA and arachidonoyl (C20:4)-CoA as acyl donors and membrane fractions of mouse tissues (liver and cerebrum, B) and culture cells (HeLa and HEK293A, C) as enzyme sources. Data are shown as the mean ± SD of three data points. The data are representative of two independent experiments with similar results.

Article Snippet: 13 C 16 Palmitoyl ( 13 C 16 16:0) coenzyme A and 13 C 18 stearoyl ( 13 C 18 18:0) coenzyme A were purchased from Taiyo Nippon Sanso (Tokyo, Japan).

Techniques: Membrane

Journal: Journal of Lipid Research

Article Title: Identification and characterization of LPLAT7 as an sn -1-specific lysophospholipid acyltransferase

doi: 10.1016/j.jlr.2022.100271

Figure Lengend Snippet: siRNA screening for sn -1 LPLATs. HeLa and HEK293A cells were treated with either siRNA for each LPLAT (LPLAT1 (AGPAT1), LPLAT2 (AGPAT2), LPLAT3 (AGPAT3), LPLAT4 (AGPAT4), LPLAT5 (AGPAT5), LPLAT6 (LCLAT1), LPLAT7 (LPGAT1), LPLAT8 (LPCAT1), LPLAT9 (LPCAT2), LPLAT10 (LPCAT4), LPLAT11 (MBOAT7), LPLAT13 (MBOAT2), and LPLAT14 (MBOAT1) or negative control siRNA. The resulting membrane fractions were used for sn -1 LPLAT assays using sn -2-rich LPC (oleoyl (C18:1) LPC, Fig. 1 A) as an acyl acceptor and stearoyl-CoA (C18:0-CoA, A: HeLa, B: HEK293A) and palmitoyl-CoA (C16:0-CoA, C: HeLa, D: HEK293A) as acyl donors. Data are shown as the mean ± SD of three data points. The data are representative of two independent experiments with similar results.

Article Snippet: 13 C 16 Palmitoyl ( 13 C 16 16:0) coenzyme A and 13 C 18 stearoyl ( 13 C 18 18:0) coenzyme A were purchased from Taiyo Nippon Sanso (Tokyo, Japan).

Techniques: Negative Control, Membrane

Journal: Journal of Lipid Research

Article Title: Identification and characterization of LPLAT7 as an sn -1-specific lysophospholipid acyltransferase

doi: 10.1016/j.jlr.2022.100271

Figure Lengend Snippet: Biochemical characterization of LPLAT7. A, B, Preference of LPLAT7 for sn -2 lysophospholipids. A: LPLAT activities of LPLAT7 against LPC. Oleoyl (C18:1) LPCs (both sn -2-rich and sn -1-rich, 20 μM) and stearoyl (C18:0)-CoA (4 μM) were used as acyl acceptors and an acyl donor, respectively. Membrane fractions from HEK293A cells transfected with a human LPLAT7 expression vector or an empty vector (Mock) were used as enzyme sources. B: Kinetic analyses of LPTAT7 activities. Substrate concentration dependence of LPLAT7 was determined with the indicated concentrations of LPCs (both sn -2-rich and sn -1-rich) in the presence of C18:0-CoA (4 μM). C, D, Determination of the glycerol sn positions into which LPLAT7 introduces a fatty acid. C: LPLAT activities of LPLAT7 against LPC using a set of labeled LPCs and a labeled-acyl-CoA. Palmitoyl (C16:0)- d 31 LPCs (both sn -2-rich and sn -1-rich, 10 μM) and 13 C 18 18:0-CoA (2 μM) were used as acyl acceptors and an acyl donor, respectively. D: The ratio of two labeled fatty acids at the sn -1 position of the LPLAT7 reaction products. The reaction products of LPLAT7 in (C) were subjected to a PLA 2 reaction. The amount of the resulting two sn -1-acyl LPCs (1– 13 C 18 18:0-2-hydroxy-GPC and 1-16:0- d 31 -2-hydroxy-GPC) was determined by LC-MS/MS. E: LPLAT activities of LPLAT7 toward acyl acceptors with different head groups. Various sn -2-rich C18:1 lyso-PLs (LPC, LPE, LPS, LPG and LPA) and soy LPI, each 20 μM and C18:0-CoA (4 μM) were used. F: Kinetic analyses of LPTAT7 activities. Substrate concentration dependence of LPLAT7 was determined with the indicated concentrations of LPCs or LPEs in the presence of C18:0-CoA (4 μM). G: LPLAT activities of LPLAT7 toward the three acyl donors, determined using C16:0-CoA, C18:1-CoA, or C18:0-CoA (4 μM) with sn -2-rich C18:1 LPC (20 μM). H: Kinetic analyses of LPTAT7 activities. Substrate concentration dependence of LPLAT7 was determined with the indicated concentrations of acyl-CoAs (C18:0-, C18:1- and C16:0-CoAs) in the presence of sn -2-rich C18:1 LPC (20 μM). I: LPLAT activities of LPLAT7 toward various acyl acceptors with different fatty acids. Several LPCs (C16:0, C18:1, linoleoyl (C18:2), arachidonoyl (C20:4), and docosahexaenoyl (C22:6), each 20 μM) and C18:0-CoA (4 μM) were used.

Article Snippet: 13 C 16 Palmitoyl ( 13 C 16 16:0) coenzyme A and 13 C 18 stearoyl ( 13 C 18 18:0) coenzyme A were purchased from Taiyo Nippon Sanso (Tokyo, Japan).

Techniques: Membrane, Transfection, Expressing, Plasmid Preparation, Concentration Assay, Labeling, Liquid Chromatography with Mass Spectroscopy

Journal: Journal of Lipid Research

Article Title: Identification and characterization of LPLAT7 as an sn -1-specific lysophospholipid acyltransferase

doi: 10.1016/j.jlr.2022.100271

Figure Lengend Snippet: LPLAT7 is the major sn -1 LPLAT in the mouse liver and human culture cells. (A, B and C) Membrane fractions of the liver from wild-type (green bars), Lplat7 heterozygous (blue bars) and Lplat7 homozygous (red bars) mice were tested for sn -1 LPLAT assays using various sn -2-rich oleoyl (C18:1) lysophospholipids (except for soy LPI) as acyl acceptors and stearoyl (C18:0)-CoA (A), palmitoyl (C16:0)-CoA (B) or oleoyl (C18:1)-CoA (C) as acyl donors. The relative LPLAT activities of wild-type mice being 100% are shown. Data are shown as the mean ± SD of 4–5 mice sample for each group. The data are representative of two independent experiments with similar results. (D, E and F) Membrane fractions of the HEK293 A parent cells (wild-type, black bars), LPLAT7 KO clone#20 (light blue bars), LPLAT7 KO clone#58 (orange bars) and LPLAT7 KO clone#116 (purple bars) were measured for sn -1 LPLAT assays using various sn -2-rich C18:1 lysophospholipids (except for soy LPI) as acyl acceptors and C18:0-CoA (D) or C16:0-CoA (E) or 18:1-CoA (F) as acyl donors. The relative LPLAT activities of parent cells (wild-type) being 100% are shown. Data are shown as the mean ± SD of three data points. The data are representative of two independent experiments with similar results. Statistically significant differences (WT vs. HT and WT vs. KO or Parent vs. KO #20, KO #58 and KO #116) are marked with asterisks indicating P -values. ∗ P <0.05; ∗∗ P <0.01; ∗∗∗ P <0.001; ∗∗∗∗ P <0.0001; ns indicates not significant; two-way ANOVA, Bonferroni’s multiple comparison test.

Article Snippet: 13 C 16 Palmitoyl ( 13 C 16 16:0) coenzyme A and 13 C 18 stearoyl ( 13 C 18 18:0) coenzyme A were purchased from Taiyo Nippon Sanso (Tokyo, Japan).

Techniques: Membrane, Comparison