Journal: Cell reports
Article Title: Triglycerides Promote Lipid Homeostasis during Hypoxic Stress by Balancing Fatty Acid Saturation.
doi: 10.1016/j.celrep.2018.08.015
Figure Lengend Snippet: Figure 2. TGs Promote Cell Viability in Low O2 and Serum by Absorbing FA Saturation (A) Viability of A498 cells expressing inducible shRNA against DGAT1 and DGAT2 mRNAs (DGAT shRNA), assessed after 72 hr under the indicated conditions (hypoxia = 0.5% O2; serum deprivation = low serum, 0.5% fetal bovine serum [FBS]) by Annexin-propidium iodide (PI) flow cytometry assay. (B) Viability of cells expressing inducible DGAT shRNAs after 72 hr under the indicated conditions (SCDi, 1 mM CAY10566) by Annexin-PI assay using flow cytometry. (C) Volcano plot showing fold change and significance of alterations in the lipidome of A498 cells cultured in low (0.5%) versus high (5%) serum. Lipids with R 1.5 fold change and p % 0.05 are displayed in color to denote lipid class. (D) Changes in FA composition or saturation of TGs, calculated by aggregating TG abundances for species containing 0, 1, or 2+ SFA chains separately. Values are normalized to control conditions (5% serum). (E) Lipid class-specific saturation indices (defined by (palmitate + stearate) / oleate) for A498 cells cultured under hypoxic (0.5% O2) versus normoxic conditions (both in low serum). (F) As (E) but with pharmacological SCD inhibition (1 mM CAY10566) instead of hypoxia. (G) Effect of serum deprivation and DGAT shRNA on total TG abundances. (H) Changes in FA makeup of TGs following DGAT knockdown; values were calculated by aggregating TG abundances for species containing 0, 1, or 2+ SFA chains separately. Values were normalized to the control condition (vehicle [Veh] treatment).
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies DGAT1 Abcam ab54037; RRID: AB_869453 V5 Life Technologies R960-25; RRID: AB_2556564 KI67 Abcam ab15580; RRID: AB_443209 Cleaved Caspase3 Cell Signaling 9661; RRID: 2341188 Calnexin Cell Signaling 2679; RRID: 10827903) Chemicals, Peptides, and Recombinant Proteins DMEM Life Technologies 11965-084 Pen/Strep Life Technologies 15140-122 Standard FBS Gemini 900-108 [U13C]-oleate Sigma 490431 T863 DGAT1i Sigma SML0539-5MG Matrigel Basement Membrane Matrix Corning 356234 200mg/kg doxycycline chow Harlan Labs TD04104 oleic acid- BSA mix Sigma O3008 Butylated hydroxytoluene (BHT) Sigma W218405 SPLASH lipidomix internal standard mix Avanti Polar Lipids 330707 Atglistatin Sigma SML1075 CAY10499 Cayman chemicals 10007875 JJKK048 Tocris 5206 Critical Commercial Assays Volupac Sartorius 11729265 RNAeasy purification kit QIAGEN 74106 High Capacity RNA-to-cDNA master mix Life Technologies 4387406 TBP Taqman assay Life Technologies HS00427620_M1 ACTB Taqman assay Life Technologies HS01060665_G1 DGAT1 Taqman assay Life Technologies HS01017541_M1 DGAT2 Taqman assay Life Technologies HS01045913_M1 QiaPrep Miniprep kit QIAGEN 27104 BODIPY 493/503 Life Technologies D3922 FITC–Annexin V, PI Kit BD Biosciences 556547 Annexin-V binding buffer BD Biosciences 556454 Deposited Data In vivo microarray study NCBI GEO GSE117774 In vitro microarray study NCBI GEO GSE117775 Experimental Models: Cell Lines A498 ATCC HTB-44 786-O ATCC CRL-1932 Experimental Models: Organisms/Strains NIH-III nude mice (female) 4-6 weeks old Charles River #201 Oligonucleotides DGAT2 Crispr1: This paper N/A Forward: caccgTGTGCTCTACTTCACTTGGC Reverse: aaacGCCAAGTGAAGTAGAGCACA (Continued on next page) e1 Cell Reports 24, 2596–2605.e1–e5, September 4, 2018
Techniques: Expressing, shRNA, Cytometry, Cell Culture, Control, Inhibition, Knockdown
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![Figure 2. TGs Promote Cell Viability in Low O2 and Serum by Absorbing FA Saturation (A) Viability of <t>A498</t> cells expressing inducible shRNA against DGAT1 and DGAT2 mRNAs (DGAT shRNA), assessed after 72 hr under the indicated conditions (hypoxia = 0.5% O2; serum deprivation = low serum, 0.5% fetal bovine serum [FBS]) by Annexin-propidium iodide (PI) flow cytometry assay. (B) Viability of cells expressing inducible DGAT shRNAs after 72 hr under the indicated conditions (SCDi, 1 mM CAY10566) by Annexin-PI assay using flow cytometry. (C) Volcano plot showing fold change and significance of alterations in the lipidome of A498 cells cultured in low (0.5%) versus high (5%) serum. Lipids with R 1.5 fold change and p % 0.05 are displayed in color to denote lipid class. (D) Changes in FA composition or saturation of TGs, calculated by aggregating TG abundances for species containing 0, 1, or 2+ SFA chains separately. Values are normalized to control conditions (5% serum). (E) Lipid class-specific saturation indices (defined by (palmitate + stearate) / oleate) for A498 cells cultured under hypoxic (0.5% O2) versus normoxic conditions (both in low serum). (F) As (E) but with pharmacological SCD inhibition (1 mM CAY10566) instead of hypoxia. (G) Effect of serum deprivation and DGAT shRNA on total TG abundances. (H) Changes in FA makeup of TGs following DGAT knockdown; values were calculated by aggregating TG abundances for species containing 0, 1, or 2+ SFA chains separately. Values were normalized to the control condition (vehicle [Veh] treatment).](https://pub-med-unpaywalled-images-cdn.bioz.com/pub_med_ids_ending_with_4495/pm30184495/pm30184495__page5_image1.jpg)
