Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Polyunsaturated fatty acid biosynthesis pathway determines ferroptosis sensitivity in gastric cancer

doi: 10.1073/pnas.2006828117

Figure Lengend Snippet: ELOVL5 and FADS1 expressions are up-regulated in mesenchymal-type GCs. (A) Fold changes in the expression of genes associated with lipid and iron metabolism in mesenchymal-type GCs (Hs746T, SNU484, SNU-668, and YCC-16 cells) compared with intestinal-type GCs (MKN-45, NCI-N87, SNU-601, and SNU-719 cells) based on the results of a microarray analysis. Volcano plot showing fold changes and P values of mRNA expression levels. Significantly up-regulated or down-regulated genes (P value <0.001, |fold change| > 3) are shown in red or blue, respectively. (B) Levels of the ELOVL2, ELOVL4, ELOVL5, FADS1, FADS2, and ACSL4 mRNAs in GCs were analyzed using qRT-PCR. Relative expression levels were normalized to the β-actin expression levels. Data are the means ± SD (n = 3 independent experiments). (C) Scheme showing the incorporation of LA into the n-6 PUFA synthesis pathway. LA, linoleic acid; GLA, gamma-linoleic acid; EDA, eicosadienoic acid; DGLA, dihomo-γ-linolenic acid; AA, arachidonic acid; AdA, adrenic acid. (D) Western blots showing the levels of ELOVL5, FADS1, ACSL4, and GPX4 proteins in mesenchymal- and intestinal-type GCs. For the detection of ELOVL5 protein, samples were not boiled. (E) Volcano plot showing fold changes and P values for lipid species in mesenchymal-type GCs and intestinal-type GCs. Free AA, AdA, and PE (18:0/22:4) are highlighted in red. (F) Levels of PUFAs and PE detected in GCs using LC-MS/MS. Intensities were normalized to the total sum of the peak areas. Data are the means ± SD (n = 5 independent experiments), with *P < 0.05 and ***P < 0.001 compared to intestinal-type cells (n = 20) using one-sided Wilcoxon rank-sum test (n.s. denotes not significant). (G) Relative amounts of labeled (m + 18) and unlabeled (m + 0) PUFAs and PE in NCI-N87, SNU-719, Hs746T, and YCC-16 cells cultured in medium containing charcoal-stripped FBS and [U-13C18] LA for 5 d.

Article Snippet: The whole-cell extracts were subjected to Western blot analysis using the following antibodies: anti-β-actin (A5316, Sigma-Aldrich), anti-FADS1 (HPA042705, Atlas Antibodies; ab126706, Abcam; sc-134337, Santa Cruz Biotechnology; GTX114528, Genetex; 10627-1-AP, Proteintech), anti-ELOVL5 (ab205535, Abcam; sc-374138, Santa Cruz Biotechnology), anti-GPX4 (ab41789, Abcam), and anti-ACSL4 (sc-271800, Santa Cruz Biotechnology).

Techniques: Expressing, Microarray, Quantitative RT-PCR, Western Blot, Liquid Chromatography with Mass Spectroscopy, Labeling, Cell Culture

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Polyunsaturated fatty acid biosynthesis pathway determines ferroptosis sensitivity in gastric cancer

doi: 10.1073/pnas.2006828117

Figure Lengend Snippet: The down-regulation of ELOVL5 and FADS1 expression alleviates ferroptosis. (A and B) Relative viability and lipid peroxidation levels in WT, ELOVL5-, and FADS1-KO YCC-16 cells treated with RSL3. Data are the means ± SD (n = 3 independent experiments, with ***P < 0.001 according to two-sided Student’s t tests). (C) Relative amounts of labeled (m + 18) and unlabeled (m + 0) PUFAs and PE in WT, ELOVL5-, and FADS1-KO YCC-16 cells cultured in medium containing charcoal-stripped FBS and [U-13C18] LA. (D) Bar plots showing the ratios of AdA (C22:4) to AA (C20:4) and AA (C20:4) to DGLA (C20:3) in ELOVL5- and FADS1-KO YCC-16 cells. Levels of PUFAs and PE in ELOVL5- and FADS1-KO YCC-16 cells determined using LC-MS/MS. Intensities were normalized to the cellular protein level. Data are the means ± SD (n = 5 independent experiments), with *P < 0.05, **P < 0.01 and ***P < 0.001 according to a two-sided Student’s test (n.s. denotes not significant).

Article Snippet: The whole-cell extracts were subjected to Western blot analysis using the following antibodies: anti-β-actin (A5316, Sigma-Aldrich), anti-FADS1 (HPA042705, Atlas Antibodies; ab126706, Abcam; sc-134337, Santa Cruz Biotechnology; GTX114528, Genetex; 10627-1-AP, Proteintech), anti-ELOVL5 (ab205535, Abcam; sc-374138, Santa Cruz Biotechnology), anti-GPX4 (ab41789, Abcam), and anti-ACSL4 (sc-271800, Santa Cruz Biotechnology).

Techniques: Expressing, Labeling, Cell Culture, Liquid Chromatography with Mass Spectroscopy

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Polyunsaturated fatty acid biosynthesis pathway determines ferroptosis sensitivity in gastric cancer

doi: 10.1073/pnas.2006828117

Figure Lengend Snippet: Inhibition of desaturase activity by SC-26196 or CP-24879 ameliorates ferroptosis. (A and B) Relative cell viability and LDH levels in Hs746T cells pretreated with 5 μM FADS2 inhibitor (SC-26196) or FADS1/2 inhibitor (CP-24879) for 4 h and treated with RSL3 for 24 h. Data are the means ± SD (n = 3 independent experiments, with **P < 0.01 according to two-sided Student’s t tests). (C) Lipid peroxidation levels in Hs746T cells pretreated with 5 μM FADS2 inhibitor (SC-26196) or FADS1/2 inhibitor (CP-24879) for 4 h and treated with RSL3 for 1 h. Data are the means ± SD (n = 3 independent experiments, with ***P < 0.001 according to two-sided Student’s t tests). (D) Cell death determined by LDH release from ELOVL5- or FADS1-depleted Hs746T cells cultured in cysteine/methionine-deficient medium for 24 h. Data are the means ± SD (n = 3 independent experiments, with *P < 0.05, **P < 0.01 and ***P < 0.001 according to two-sided Student’s t tests). (E) Cell death measured by LDH release from Hs746T cells pretreated with FADS inhibitors for 4 h, followed by an incubation with cysteine/methionine-deficient medium for 24 h. Data are the means ± SD (n = 3 independent experiments, with **P < 0.01 and ***P < 0.001 according to two-sided Student’s t tests).

Article Snippet: The whole-cell extracts were subjected to Western blot analysis using the following antibodies: anti-β-actin (A5316, Sigma-Aldrich), anti-FADS1 (HPA042705, Atlas Antibodies; ab126706, Abcam; sc-134337, Santa Cruz Biotechnology; GTX114528, Genetex; 10627-1-AP, Proteintech), anti-ELOVL5 (ab205535, Abcam; sc-374138, Santa Cruz Biotechnology), anti-GPX4 (ab41789, Abcam), and anti-ACSL4 (sc-271800, Santa Cruz Biotechnology).

Techniques: Inhibition, Activity Assay, Cell Culture, Incubation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Polyunsaturated fatty acid biosynthesis pathway determines ferroptosis sensitivity in gastric cancer

doi: 10.1073/pnas.2006828117

Figure Lengend Snippet: ELOVL5 and FADS1 expression is down-regulated through DNA hypermethylation. (A and B) Manhattan plot of the methylation levels and statistical significance of methylation at each CpG site in the promoter regions of ELOVL5 (A) and FADS1/2 (B) in mesenchymal-type (Hs746T, SNU484, SNU-668, and YCC-16) and intestinal-type (NCI-N87, SNU-719, SNU-601, and MKN-45) GCs. The putative enhancer/promoter region of ELOVL5 (chr6: 53,211,316 to 53,214,820) is highlighted in orange. The putative regions of the FADS1 promoter (chr11: 61,584,650 to 61,586,300), FADS2 promoter (chr11: 61,594,300 to 61,595,600) and putative enhancer (chr11: 61,587,300 to 61,589,000) are colored in green, blue, and orange, respectively (36).

Article Snippet: The whole-cell extracts were subjected to Western blot analysis using the following antibodies: anti-β-actin (A5316, Sigma-Aldrich), anti-FADS1 (HPA042705, Atlas Antibodies; ab126706, Abcam; sc-134337, Santa Cruz Biotechnology; GTX114528, Genetex; 10627-1-AP, Proteintech), anti-ELOVL5 (ab205535, Abcam; sc-374138, Santa Cruz Biotechnology), anti-GPX4 (ab41789, Abcam), and anti-ACSL4 (sc-271800, Santa Cruz Biotechnology).

Techniques: Expressing, Methylation

Journal: Scientific Reports

Article Title: Expression of genes and localization of enzymes involved in polyunsaturated fatty acid synthesis in rabbit testis and epididymis

doi: 10.1038/s41598-022-06700-y

Figure Lengend Snippet: Expression of elongase 2 and 5 (ELOVL2 and ELOVL5) and Δ5- and Δ6-desaturase (FADS1 and FADS2) in rabbit testis. CNT: pointed bar; FLAX: full grey bar. *Significance of diet; comparison by t-test ( p < 0.05).

Article Snippet: Specimens were treated overnight at 4 °C with the primary antibodies anti-FADS1 (Δ5-desaturase; Sigma-Aldrich, St. Louis, MO, USA) diluted 1 : 20, anti-FADS2 (Δ6-desaturase; Sigma-Aldrich, St. Louis, MO, USA) diluted 1 : 20, anti-ELOVL5 (elongase 5; Sigma-Aldrich, St. Louis, MO, USA) diluted 1 : 100, anti-ELOVL2 (elongase 2; Sigma-Aldrich, St. Louis, MO, USA) diluted 1 : 70, anti-DHA, anti-FITC-linked EPA and anti-PE-linked ARA (MyBioSource Inc., San Diego, CA, USA) diluted 1 : 50.

Techniques: Expressing

Journal: Scientific Reports

Article Title: Expression of genes and localization of enzymes involved in polyunsaturated fatty acid synthesis in rabbit testis and epididymis

doi: 10.1038/s41598-022-06700-y

Figure Lengend Snippet: Immunolocalization of Δ6-desaturase (FADS2) in testis ( a , b ) and epididymis ( c , d ); very long-chain fatty acid 5 elongase (ELOVL5) in testis ( e , f ) and epididymis ( g , h ); Δ5-desaturase (FADS1) in testis ( i , j ) and epididymis ( k , l ); and very long-chain fatty acid 2 elongase (ELOVL2) in testis ( m , n ) and epididymis ( o , p ). A high labelling intensity was evident in the interstitial tissue in testis from rabbits fed control ( a ) and FLAX diets ( b ); in the latter, the FADS2 signal was present also in elongated spermatids (arrow). In the cauda epididymis (control c , FLAX d ), FADS2 was absent from the principal cells; FLAX diet increased the presence of the enzyme in interstitial connective tissue. A high labelling intensity of ELOVL5 was shown in spermatocytes and round spermatids in testis from rabbits fed control ( e ) and FLAX diets ( f ). In cauda epididymis control and FLAX ( g , h respectively), ELOVL5 appeared in principal epidydimal cells, in interstitial connective tissue and in numerous vesicles in the lumen where the spermatozoa were located. A clear signal of FADS1 was detected in Sertoli cells in testis from rabbits fed control ( i ) and FLAX diets ( j ); in the latter, the fluorescence intensity was increased and present in elongated spermatids. In the cauda epididymis, both control and FLAX ( k , l ), FADS1 was localized in interstitial connective tissue, basal and principal epididymal cells, and in a very large number of epididymal vesicles. A high labelling intensity of ELOVL2 was evident in the interstitial tissue in testis from rabbits fed control ( m ) and FLAX diets ( n ). In the cauda epididymis ( o , p ), ELOVL2 appeared localized in basal and principal epididymal cells as well as in interstitial connective tissue; a small number of vesicles appeared fluorescent in the lumen where the spermatozoa were located (more evident in FLAX diet). Bar: 50 µm.

Article Snippet: Specimens were treated overnight at 4 °C with the primary antibodies anti-FADS1 (Δ5-desaturase; Sigma-Aldrich, St. Louis, MO, USA) diluted 1 : 20, anti-FADS2 (Δ6-desaturase; Sigma-Aldrich, St. Louis, MO, USA) diluted 1 : 20, anti-ELOVL5 (elongase 5; Sigma-Aldrich, St. Louis, MO, USA) diluted 1 : 100, anti-ELOVL2 (elongase 2; Sigma-Aldrich, St. Louis, MO, USA) diluted 1 : 70, anti-DHA, anti-FITC-linked EPA and anti-PE-linked ARA (MyBioSource Inc., San Diego, CA, USA) diluted 1 : 50.

Techniques: Fluorescence

Journal: Scientific Reports

Article Title: Expression of genes and localization of enzymes involved in polyunsaturated fatty acid synthesis in rabbit testis and epididymis

doi: 10.1038/s41598-022-06700-y

Figure Lengend Snippet: Schematic representation of enzyme and fatty acid localization in epididymal cells. ELOVL2: elongase 2; ELOVL5: elongase 5; FADS1: Δ5-desaturase; FADS2: Δ6-desaturase; DHA: docosahexaenoic acid; EPA: eicosapentaenoic acid; ARA: arachidonic acid. In the connective tissue of cauda epididymis all enzymes were expressed (FADS1 and 2, ELOVL2 and 5), while in the basal cell only FADS1 and ELOV2 were found and in the principal cell ELOVL5, 2 and FADS1. Conversely the FAs localization was similar (ARA, DHA and EPA in both cells). Furthermore, in the epididymal vesicles, the expression of ELOVL5,2 and FADS 1 was found.

Article Snippet: Specimens were treated overnight at 4 °C with the primary antibodies anti-FADS1 (Δ5-desaturase; Sigma-Aldrich, St. Louis, MO, USA) diluted 1 : 20, anti-FADS2 (Δ6-desaturase; Sigma-Aldrich, St. Louis, MO, USA) diluted 1 : 20, anti-ELOVL5 (elongase 5; Sigma-Aldrich, St. Louis, MO, USA) diluted 1 : 100, anti-ELOVL2 (elongase 2; Sigma-Aldrich, St. Louis, MO, USA) diluted 1 : 70, anti-DHA, anti-FITC-linked EPA and anti-PE-linked ARA (MyBioSource Inc., San Diego, CA, USA) diluted 1 : 50.

Techniques: Expressing

Journal: Scientific Reports

Article Title: Expression of genes and localization of enzymes involved in polyunsaturated fatty acid synthesis in rabbit testis and epididymis

doi: 10.1038/s41598-022-06700-y

Figure Lengend Snippet: Schematic representation of enzyme and fatty acid localization in testis cells. The localization of the enzymes studied in germ, Sertoli and Leydig cells is shown on the left, whereas the localization of fatty acids is shown on the right. ELOVL2: elongase 2; ELOVL5: elongase 5; FADS1: Δ5-desaturase; FADS2: Δ6-desaturase; DHA: docosahexaenoic acid; EPA: eicosapentaenoic acid; ARA: arachidonic acid.

Article Snippet: Specimens were treated overnight at 4 °C with the primary antibodies anti-FADS1 (Δ5-desaturase; Sigma-Aldrich, St. Louis, MO, USA) diluted 1 : 20, anti-FADS2 (Δ6-desaturase; Sigma-Aldrich, St. Louis, MO, USA) diluted 1 : 20, anti-ELOVL5 (elongase 5; Sigma-Aldrich, St. Louis, MO, USA) diluted 1 : 100, anti-ELOVL2 (elongase 2; Sigma-Aldrich, St. Louis, MO, USA) diluted 1 : 70, anti-DHA, anti-FITC-linked EPA and anti-PE-linked ARA (MyBioSource Inc., San Diego, CA, USA) diluted 1 : 50.

Techniques: