Journal: iScience

Article Title: Phospholipase PLA2G7 is complementary to GPX4 in mitigating punicic-acid-induced ferroptosis in prostate cancer cells

doi: 10.1016/j.isci.2024.109774

Figure Lengend Snippet: Phospholipase PLA2G7 is involved in the suppression of punicic-acid-induced ferroptosis in PCa cells (A) Bar plot of the mRNA expression of PL-related genes from the Expression Public 22Q4 dataset differentially expressed between 22RV1 and PC3 cells. Gene expression is expressed as log2(TMP+1) from RNA sequencing data. (B) Relative mRNA quantity of PLA2G10, PLA2G6, and PLA2G7 in 22RV1 and PC3 cells, expressed as 2 −ΔCt with the geometric mean of the Ct of three reference genes (i.e., GUSB, TBP, and β-ACTIN). Data points represent individual RT-qPCR technical replicates ( N = 3, n = 3). (C) Boxplot of the expression of PLA2G7 in normal prostate and prostate tumor tissues from patients of the TCGA cohort. Gene expression is expressed as log2(TMP+1). The Limma package was used for Student’s t test comparing PLA2G7 mean expression in normal versus tumor tissues. Each data point represents a patient sample. (D) Fold change in PLA2G7 gene expression in 22RV1 and PC3 cells treated for 24 h with punicic acid (PunA) or α-linolenic acid (ALA) 1.5 μM, normalized to the control (untreated cells). Data points represent individual RT-qPCR technical replicates ( N = 3, n = 3). (E) Ratio of PLA2G7-to-valosin-containing protein (VCP) protein abundance in 22RV1 and PC3 cells treated with either no fatty acid (FA) (control), PunA 1.5 μM, or ALA 1.5 μM for 24 h. Representative western blot is shown below. (F) Relative viability of 22RV1 cells after treatment with either vehicle (DMSO 0.1% v/v, control), Darapladib 0.1 μM (Dara), PunA 17.75 μM, or a combination thereof for 24 h. Data were normalized to the control. (G) Fold change in PLA2G7 gene expression of 22RV1 cells transfected with either a negative control siRNA (siNeg) or a siRNA pool targeting PLA2G7 (siPLA2G7), normalized to siNeg. Data points represent individual RT-qPCR technical replicates ( N = 3, n = 3). (H) Relative viability of siNeg and siPLA2G7 22RV1 cells treated for 24 h with the indicated doses of PunA, normalized to the control (untreated cells). (I) Fold change in PLA2G7 gene expression of PC3 cells overexpressing either a negative control vector (OE-CTL) or PLA2G7 (OE-PLA2G7), normalized to OE-CTL. Data points represent individual RT-qPCR technical replicates ( N = 3, n = 3). (J) Relative viability of OE-CTL and OE-PLA2G7 PC3 cells treated with the indicated doses of PunA for 24 h, normalized to the control (untreated cells). (K) Lipid peroxidation levels in OE-CTL and OE-PLA2G7 PC3 cells after 4 h of treatment with the indicated doses of PunA. Data are expressed as the fold change in the green-to-red fluorescence ratio of C11-BODIPY and normalized to the control. (L) Representative thin-layer chromatography plate profile of PLOOH species after incubation for 0, 15, 30, or 45 min in the presence of PLA2G7 recombinant enzyme. A control without PLOOH was also added on the plate (corresponding to [−]). Data are represented as mean ± SEM of N = 3 independent cultures (D–K) or as median with the 25–75 percentiles of nine replicates (B) or of 552 individual patient samples (C). Significance was established by Student’s t test (B, G, H, J, K), one-way ANOVA with Sidak’s multiple comparisons (D, F), or two-way ANOVA with Sidak’s multiple comparisons (E, I, L, M). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. For two-way ANOVA comparing both treatments and cell lines or clones, statistical significance is indicated by letters, with capital letters (A) for comparing different treatments for the same cell line/clone (e.g., siNeg) and small letters (a) for comparing cell lines/clones for the same treatment (e.g., siNeg versus siPLA2G7). See also Figure S6 .

Article Snippet: Darapladib , Selleck chemicals , Cat# S7520.

Techniques: Expressing, Gene Expression, RNA Sequencing, Quantitative RT-PCR, Control, Quantitative Proteomics, Western Blot, Transfection, Negative Control, Plasmid Preparation, Fluorescence, Thin Layer Chromatography, Incubation, Recombinant, Clone Assay

Journal: iScience

Article Title: Phospholipase PLA2G7 is complementary to GPX4 in mitigating punicic-acid-induced ferroptosis in prostate cancer cells

doi: 10.1016/j.isci.2024.109774

Figure Lengend Snippet: Phospholipase PLA2G7 acts complementary to GPX4 to protect PCa cells from punicic-acid-triggered ferroptosis (A) Relative viability of 22RV1 cells treated with either vehicle (DMSO 0.1% v/v, control), punicic acid (PunA) 3 μM, Ras-selective lethal (RSL3) 0.1 μM, Darapladib (Dara) 0.1 μM, or combinations thereof for 24 h, normalized to the control. (B) Relative viability of 22RV1 cells transfected with either a negative control siRNA (siNeg) or a siRNA pool targeting GPX4 (siGPX4), treated for 24 h with vehicle (DMSO 0.1% v/v, control), PunA 10 μM, Dara 0.1 μM, or a combination thereof, normalized to the control. (C) Lipid peroxidation levels in siNeg and siGPX4 22RV1 cells after 4 h of treatment with vehicle (DMSO 0.1% v/v, control), PunA 10 μM, Dara 0.1 μM, or a combination thereof. Data are expressed as the fold change of the green-to-red fluorescence ratio of C11-BODIPY and normalized to the control. (D) Relative viability of 22RV1 cells transfected with either a negative control siRNA (siNeg) or a siRNA pool targeting PLA2G7 (siPLA2G7) treated for 24 h with vehicle (DMSO 0.1% v/v, control), PunA 50 μM, RSL3 0.3 μM, or a combination thereof, normalized to the control. (E) Lipid peroxidation levels in siNeg and siPLA2G7 22RV1 cells after 4 h of treatment with vehicle (DMSO 0.1% v/v, control), PunA 50 μM, RSL3 0.3 μM, or a combination thereof. Data are expressed as the fold change of the green-to-red fluorescence ratio of C11-BODIPY and normalized to the control. (F) Relative viability of PC3 cells overexpressing either a negative control vector (OE-CTL) or PLA2G7 (OE-PLA2G7) and transfected with either a negative control vector (-CTL) or a GPX4-expressing vector (-GPX4), treated for 24 h with the indicated doses of PunA, normalized to the control (untreated cells). (G) Lipid peroxidation levels in OE-CTL-CTL, OE-PLA2G7-CTL, OE-CTL-GPX4, and OE-PLA2G7-GPX4 PC3 cells treated for 4 h with the indicated doses of PunA. Data are expressed as the fold change of the green-to-red fluorescence ratio of C11-BODIPY and normalized to the control (untreated cells). (H) Correlation between the cell resistance score and the PunA IC50 of PC3, DU145, LNCaP, C4-2B, VCaP, and 22RV1 cell lines. Cell resistance score was defined as the product of the abundances of GPX4 and PLA2G7 proteins over the one of ACSL4 protein, determined by western blot and normalized to β-ACTIN (for GPX4 and ACSL4) or valosin-containing protein (VCP) (for PLA2G7) protein abundance. Data are represented as mean ± SEM of N ≥ 3 independent cultures (A–H). Significance was established by one-way ANOVA with Sidak’s multiple comparisons (A) or two-way ANOVA with Sidak’s multiple comparisons (B–G). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. For two-way ANOVA comparing both treatments and cell clones, statistical significance is indicated by letters, with capital letters (A) for comparing different treatments for the same clone (e.g., OE-CTL-CTL) and small letters (a) for comparing clones for the same treatment (e.g., OE-CTL-CTL vs. OE-CTL-GPX4). See also Figure S7 .

Article Snippet: Darapladib , Selleck chemicals , Cat# S7520.

Techniques: Control, Transfection, Negative Control, Fluorescence, Plasmid Preparation, Expressing, Western Blot, Quantitative Proteomics, Clone Assay

Journal: iScience

Article Title: Phospholipase PLA2G7 is complementary to GPX4 in mitigating punicic-acid-induced ferroptosis in prostate cancer cells

doi: 10.1016/j.isci.2024.109774

Figure Lengend Snippet:

Article Snippet: Darapladib , Selleck chemicals , Cat# S7520.

Techniques: Virus, Control, Expressing, Plasmid Preparation, Recombinant, Western Blot, Transfection, Saline, Protease Inhibitor, cDNA Synthesis, Bicinchoninic Acid Protein Assay, Software, RNA Sequencing

Journal: Neural Regeneration Research

Article Title: Inhibiting ceramide synthase 5 expression in microglia decreases neuroinflammation after spinal cord injury

doi: 10.4103/nrr.nrr-d-23-01933

Figure Lengend Snippet: Figure 7 ｜Pla2g7 is essential for mediating the regulatory effects of CerS5 on pyroptosis. (A) Ceramide concentrations in the spinal cord in the SCI and Sham groups were detected by ELISA, and the data were normalized to the Shame group (n = 3). (B) Western blot analysis of Pla2g7, p50, GSDMD, and GSDMD-N expression levels in BV2 cells after treatment with Darapladib (Pla2g7 inhibitor) (n = 3). **P < 0.01, vs. C16 ceramide. (C) Representative examples of immunofluorescence staining images used to quantify GSDMD+Iba1+ cells after treatment with Darapladib (n = 5). There were significantly fewer GSDMD+Iba1+ cells in the C16 ceramide + Darapladib group than in the C16 ceramide group. Iba1, green, CoraLite 488; GSDMD, red, CoraLite 594; DAPI, blue. Scale bar: 100 μm. (D) Western blot analysis of the expression levels of proteins associated with the classical NLRP3 pyroptosis pathway in BV2 cells after treatment with siCerS5 or Darapladib (n = 3). Data are expressed as the mean ± SEM. *P < 0.05, **P < 0.01 (one-way analysis of variance followed by Tukey’s multiple comparison test). ASC: Apoptosis associated speck like protein containing CARD; CerS: ceramide synthase; DAPI: 4′,6-diamidino-2-phenylindole; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GSDMD: gasdermin D; GSDMD-N: N-terminal GSDMD; Iba1: ionized calcium binding adaptor molecule 1; NLRP3: NOD-like receptor thermal protein domain associated protein 3; SCI: spinal cord injury; si.CerS5: CerS5 siRNA.

Article Snippet: The mice were randomly assigned to one of five groups (10 mice per group): (1) Sham group, with no SCI induced after opening the vertebral plate; (2) SCI group, with no intrathecal injection after striking the spinal cord; (3) SCI + si.CerS5 group, which were injected with 10 nmol CerS5 siRNA (si.CerS5) (synthesized by GenePharm, Shanghai, China; Table 1) intrathecally around the notch immediately after contusion; (4) SCI + si.NC group, which were injected intrathecally with si.NC (10 nmol) (synthesized by GenePharm, Shanghai, China; Table 1) around the notch immediately after contusion; (5) SCI + Darapladib group, which were injected intrathecally with Darapladib (a Pla2g7 inhibitor, 10 mg/kg, TargetMol, T6109, Boston, MA, USA) around the notch immediately after contusion; and (6) SCI + Vehicle group, which were injected intrathecally with vehicle (10% dimethylsulfoxide, 40% polyethylene glycol 300, 5% Tween-80, and 45% phosphate-buffered saline) around the notch immediately after contusion.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Immunofluorescence, Staining, Comparison, Binding Assay

Journal: Neural Regeneration Research

Article Title: Inhibiting ceramide synthase 5 expression in microglia decreases neuroinflammation after spinal cord injury

doi: 10.4103/nrr.nrr-d-23-01933

Figure Lengend Snippet: Figure 8 ｜Darapladib inhibits microglial pyroptosis in the spinal cord after SCI. (A) Representative examples of immunofluorescence staining images used to quantify GSDMD+Iba1+ cells in the SCI and Sham groups after treatment with Darapladib (n = 5). There were significantly fewer GSDMD+Iba1+ cells in the SCI + Darapladib group compared with the SCI and SCI + Vehicle groups. Iba1, green, CoraLite 488; GSDMD, red, CoraLite 594; DAPI, blue. Scale bars: 100 μm (upper); 20 μm (lower). (B) Western blot analysis of the expression levels of Pla2g7 and proteins associated with the classical NLRP3 pyroptosis pathway in the SCI and Sham groups after treatment with Darapladib (n = 3). Data are expressed as the mean ± SEM. *P < 0.05, **P < 0.01, vs. SCI group (one-way analysis of variance followed by Tukey’s multiple comparison test). ASC: Apoptosis associated speck like protein containing CARD; CerS: ceramide synthase; DAPI: 4′,6-diamidino-2-phenylindole; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GSDMD: gasdermin D; GSDMD-N: N-term gasdermin D; IL: interleukin; NLRP3: NOD-like receptor thermal protein domain associated protein 3; Pla2g7: phospholipase a2 group VII; SCI: spinal cord injury; si.CerS5: CerS5 siRNA.

Article Snippet: The mice were randomly assigned to one of five groups (10 mice per group): (1) Sham group, with no SCI induced after opening the vertebral plate; (2) SCI group, with no intrathecal injection after striking the spinal cord; (3) SCI + si.CerS5 group, which were injected with 10 nmol CerS5 siRNA (si.CerS5) (synthesized by GenePharm, Shanghai, China; Table 1) intrathecally around the notch immediately after contusion; (4) SCI + si.NC group, which were injected intrathecally with si.NC (10 nmol) (synthesized by GenePharm, Shanghai, China; Table 1) around the notch immediately after contusion; (5) SCI + Darapladib group, which were injected intrathecally with Darapladib (a Pla2g7 inhibitor, 10 mg/kg, TargetMol, T6109, Boston, MA, USA) around the notch immediately after contusion; and (6) SCI + Vehicle group, which were injected intrathecally with vehicle (10% dimethylsulfoxide, 40% polyethylene glycol 300, 5% Tween-80, and 45% phosphate-buffered saline) around the notch immediately after contusion.

Techniques: Immunofluorescence, Staining, Western Blot, Expressing, Comparison

Journal: PLoS ONE

Article Title: Pharmacogenetic meta-analysis of baseline risk factors, pharmacodynamic, efficacy and tolerability endpoints from two large global cardiovascular outcomes trials for darapladib

doi: 10.1371/journal.pone.0182115

Figure Lengend Snippet: Characteristics of PGx study subjects (n = 23,981).

Article Snippet: Darapladib (SB-480848) is a novel, selective, reversible, orally active inhibitor of Lp-PLA 2 activity that was in development by GlaxoSmithKline (GSK) for CV risk reduction.

Techniques: Activity Assay

Journal: PLoS ONE

Article Title: Pharmacogenetic meta-analysis of baseline risk factors, pharmacodynamic, efficacy and tolerability endpoints from two large global cardiovascular outcomes trials for darapladib

doi: 10.1371/journal.pone.0182115

Figure Lengend Snippet: List of significant results for efficacy endpoints (MCE and MI).

Article Snippet: Darapladib (SB-480848) is a novel, selective, reversible, orally active inhibitor of Lp-PLA 2 activity that was in development by GlaxoSmithKline (GSK) for CV risk reduction.

Techniques: Variant Assay

Journal: JAMA Cardiology

Article Title: Association of Fibroblast Growth Factor 23 With Recurrent Cardiovascular Events in Patients After an Acute Coronary Syndrome

doi: 10.1001/jamacardio.2018.0653

Figure Lengend Snippet: Covariates in the adjustment model were age, sex, current smoker, diabetes, body mass index, race/ethnicity, hypertension, hyperlipidemia, region, prior myocardial infarction (MI), index diagnosis (non–ST-elevation MI vs ST-elevation MI, unstable angina vs ST-elevation MI), low-density lipoprotein cholesterol level, days from qualifying event, randomized treatment arm, estimated glomerular filtration rate (Modification of Diet in Renal Dysfunction), high-sensitivity troponin I concentration, brain-type natriuretic peptide concentration, and high-sensitivity C-reactive protein concentration. CV indicates cardiovascular; HR, hazard ratio.

Article Snippet: Stabilization of Plaques Using Darapladib-Thrombolysis in Myocardial Infarction was sponsored by GlaxoSmithKline.

Techniques: Biomarker Discovery, Filtration, Modification, Concentration Assay, Protein Concentration

Journal: Clinical Ophthalmology (Auckland, N.Z.)

Article Title: The Evolving Treatment of Diabetic Retinopathy

doi: 10.2147/OPTH.S236637

Figure Lengend Snippet: Current Investigational Pharmacotherapy for Diabetic Retinopathy and Diabetic Macular Edema (1–4)

Article Snippet: Darapladib , Phospholipase CA2 inhibitor , PO , II , GlaxoSmithKline.

Techniques: Permeability, Inhibition, Clinical Proteomics