Journal: eLife

Article Title: Human DECR1 is an androgen-repressed survival factor that regulates PUFA oxidation to protect prostate tumor cells from ferroptosis

doi: 10.7554/eLife.54166

Figure Lengend Snippet: ( A ) Schematic of DECR1 function in fatty acid (FA) β-oxidation. In order to translocate FAs into the mitochondria, CPT1 converts long-chain acyl-CoA species to their corresponding long-chain acylcarnitine species. This is followed by a dehydrogenation step mediated by acyl CoA dehydrogenase (ACAD) to generate trans-2-enoyl-CoA, the only intermediate that can be processed by downstream enzymes in the β-oxidation process. Many FAs have unsaturated bonds either on an odd-numbered carbon or in the cis-configuration, resulting in the generation of enoyl-CoA intermediates that cannot be directly processed via the downstream β-oxidation enzymes. These FAs require the activity of 3 auxiliary enzymes, ECI1, ECH1 and DECR1 in order to form trans-2-enoyl-CoA before undergoing β-oxidation. DECR1 catalyzes the conversion of either 2-trans,4-cis-dienoyl or 2-trans,4-trans-dienoyl-CoA to 3-trans-enoyl-CoA. A complete cycle of β-oxidation results in the release of the first two carbon units as acetyl-CoA, and a fatty-acyl-CoA minus two carbons. The acetyl-CoA enters the TCA cycle to produce energy (ATP). The shortened fatty-acyl-CoA is processed again starting with the ACADs to form trans-2-enoyl-CoA either directly or with the aid of the auxiliary enzymes depending on the presence of double bonds. This process continues until all carbons in the fatty acid chain are turned into acetyl-CoA. ( B ) DECR1 protein expression after 72 hr or 96 hr siRNA transfection. Densitometry quantification of relative DECR1 protein expression was normalized to the HSP90 internal control. ( C ) Linoleic acid level in LNCaP cells quantified in following 96 hr DECR1 knockdown using GC QQQ targeted metabolomics. ( D ) Relative quantities of the C10:2 acylcarnitine species in LNCaP cell conditioned medium (left) or cell lysates (right) (n = 3). ( E ) Quantification of ATP levels in LNCaP cell lysates. LNCaP cells were transfected with DECR1 siRNAs for 48 hr and then starved in no-glucose medium and treated with the lipolysis inhibitor DEUP (100 µM) in the presence (BSA-LA) or absence (BSA) of the PUFA linoleic acid for 48 hr before measuring ATP levels. ( F ) Oxygen consumption rate (OCR) was assessed in LNCaP cells supplemented with the PUFA linoleic acid (LA) or ( G ) the saturated fatty acid palmitic acid (PA). Each data point represents an OCR measurement. ATP production, maximal mitochondrial respiration and mitochondrial spare capacity were assessed. ( H ) Extracellular acidification rate (ECAR) was assessed in LNCaP cells. Each data point represents an ECAR measurement. For experiments ( F-H ) LNCaP cells were transfected with DECR1 siRNAs for 72 hr, then starved in substrate limited medium for 24 hr; the assay was run in FAO assay medium. ( I and J ) Metabolites were quantified in LNCaP cells following 96 hr DECR1 knockdown using GC QQQ targeted metabolomics. Data in bar graphs are represented as the mean ± s.e.m (n = 3). Statistical analysis was performed using two-tailed Student’s t -test: *p<0.05, **p<0.01 and ****p<0.0001.

Article Snippet: LNCaP cells were transduced with the universal negative control shRNA lentiviral particles (shControl), DECR1 shRNA lentiviral particles (shDECR1) or hDECR1 (GFP-Puro) designed by GenTarget Inc (San Diego, CA, USA) according to the manufacturer’s protocol ( ).

Techniques: Activity Assay, Expressing, Transfection, Control, Knockdown, Two Tailed Test

Journal: eLife

Article Title: Human DECR1 is an androgen-repressed survival factor that regulates PUFA oxidation to protect prostate tumor cells from ferroptosis

doi: 10.7554/eLife.54166

Figure Lengend Snippet: ( A ) Cell viability after DECR1 knockdown in non-malignant PNT1 prostate cells; hormone-responsive PCa cell lines (LNCaP and VCaP); castrate-resistant V16D and 22RV1 cell lines and enzalutamide-resistant MR94F cells cultured in full serum media. ( B ) Cell viability and cell death of stable DECR1 knockdown LNCaP cells cultured in full serum media. ( C ) Cell viability of stable DECR1-overexpressed LNCaP cells cultured in full serum media. Cell viability and cell death were measured using trypan blue exclusion following 96 hr DECR1 knockdown. Percentages are represented relative to the control siRNA; n = 3 independent experiments per cell line. ( D ) Clonogenic cell survival of LNCaP cells was assessed using colony formation assay. Stable DECR1-overexpressed cells or ( E ) stable DECR1 knockdown was achieved using two different short hairpin (sh) vectors and DECR1 expression was confirmed using western blot. Cells were cultured for 2 weeks, washed with PBS, fixed with paraformaldehyde and stained with 1% crystal violet for 30 min. Colonies with more than 50 cells were counted manually; data shown is representative of n = 2 independent experiments. ( F ) LNCaP and 22RV1 cell growth in 3D spheres. Spheroids were prepared using the hang drop assay following 48 hr DECR1 knockdown. Spheroid volumes were determined after five days of culturing the cells in 20 µl drops; at least 25 spheres per cell line were assessed using the ReViSP software, n = 3 independent experiments per cell line. ( G ) LNCaP, 22RV1 and MR49F cell migration and ( H ) 22RV1 cell invasion were assessed using a transwell migration/invasion assay. Cells were transfected with DECR1 siRNA or control siRNA for 48 hr prior to the assay; data shown is representative of n = 3 independent experiments. ( I ) Violin plots of mKi67 and DECR1 mRNA expression in subcutaneous LNCaP tumors (n = 5 mice, shControl; n = 4 mice, shDECR1). ( J ) Representative Ki67 IHC staining of subcutaneous LNCaP tumors. Scale bar, 100 μm. Data in bar graphs are represented as the mean ± s.e.m. Statistical analysis was performed using one-way ANOVA, followed by Dunnett's multiple comparisons test: *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. ( K ) Tumor growth of intraprostatically injected LNCaP cells (shControl and shDECR1). (J) Lung luminescence readings following DECR1 knockdown in mice. Data are presented as mean ± s.e.m. Statistical analysis was performed using two-way ANOVA or two-tailed student’s t-test: *p<0.05 and ***p<0.001.

Article Snippet: LNCaP cells were transduced with the universal negative control shRNA lentiviral particles (shControl), DECR1 shRNA lentiviral particles (shDECR1) or hDECR1 (GFP-Puro) designed by GenTarget Inc (San Diego, CA, USA) according to the manufacturer’s protocol ( ).

Techniques: Knockdown, Cell Culture, Control, Colony Assay, Expressing, Western Blot, Staining, Software, Migration, Invasion Assay, Transfection, Immunohistochemistry, Injection, Two Tailed Test

Journal: eLife

Article Title: Human DECR1 is an androgen-repressed survival factor that regulates PUFA oxidation to protect prostate tumor cells from ferroptosis

doi: 10.7554/eLife.54166

Figure Lengend Snippet: ( A ) Individual tumor growth of shControl and shDECR1 cells in LNCaP-xenograft tumors. ( B ) Violin plots of mKi67 and DECR1 mRNA expression in LNCaP tumors (n = 5 mice, shControl; n = 4 mice, shDECR1). ( C ) Representative DECR1 and KI67 IHC staining of consecutive sections of LNCaP-xenograft tumors. ( D ) Individual tumor growth of shControl and shDECR1 cells in LNCaP-xenograft tumors from the second cohort of mice. ( E ) Tumor growth was monitored based on luciferase activity over time as indicated by IVIS imaging (n = 10 mice, shControl; n = 9 mice, shDECR1). Statistical analysis was performed using two-tailed Student’s t -test: *p<0.05 and ***p<0.001.

Article Snippet: LNCaP cells were transduced with the universal negative control shRNA lentiviral particles (shControl), DECR1 shRNA lentiviral particles (shDECR1) or hDECR1 (GFP-Puro) designed by GenTarget Inc (San Diego, CA, USA) according to the manufacturer’s protocol ( ).

Techniques: Expressing, Immunohistochemistry, Luciferase, Activity Assay, Imaging, Two Tailed Test

Journal: eLife

Article Title: Human DECR1 is an androgen-repressed survival factor that regulates PUFA oxidation to protect prostate tumor cells from ferroptosis

doi: 10.7554/eLife.54166

Figure Lengend Snippet: The sequence of the DECR1 shRNA and hDECR1 vectors.

Article Snippet: LNCaP cells were transduced with the universal negative control shRNA lentiviral particles (shControl), DECR1 shRNA lentiviral particles (shDECR1) or hDECR1 (GFP-Puro) designed by GenTarget Inc (San Diego, CA, USA) according to the manufacturer’s protocol ( ).

Techniques: Sequencing, shRNA