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: Mouse polyclonal anti-PLA2G6 , Santa Cruz antibody , Cat# sc376563 RRID: AB_ 11150308.

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

Journal: bioRxiv

Article Title: Microglia-Dependent and Independent Modulation of Brain Lipid Metabolism in Alzheimer’s Disease Revealed by Pharmacological and Genetic Microglial Depletion

doi: 10.1101/2024.11.18.624173

Figure Lengend Snippet: Western-blot analysis (A) of calcium-dependent and independent phospholipase A2 (phosphor-cPLA2 and iPLA2, respectively), 4HNE levels and their quantification results (B, C, and D). (E) 4-HNE level measure by MDMS-shotgun lipidomics. Gene Ontology (GO) terms analysis of genes that negatively correlated with LPE in pharmacological (F) and genetic (G) microglial elimination cohort. Data transformation: square root for the pharmacological cohort, cube root for the genetic cohort; data scaling: pareto for the pharmacological cohort, mean for the genetic cohort. All data presented as mean ± SEM, normalized to WT. Two tailed two-way ANOVA with Turkey correction, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Article Snippet: PVDF membranes (CliniSciences, Nanterre, France) with the transferred protein were incubated with primary antibodies (1:1000-2000 dilutions) of anti-6E10 (mouse, BioLegend, Inc., San Diego, California, USA), anti-Homer1 (rabbit, Cell Signaling Technology, Boston, MA, USA), anti-CD68 (E307V) (rabbit, Cell Signaling Technology, Boston, MA, USA), anti-LAMP-1 (1D4B) (rat, Thermo Fisher Scientific, Waltham, MA, USA); anti-4HNE (12F7) (mouse, Thermo Fisher Scientific, Waltham, MA, USA), anti-PGRN (sheep, R&D Systems, Minneapolis, MN, USA), anti-Phospho-PLA2G4A (Ser505) (Rabbit, Proteintech Group, Inc, Rosemont, IL, USA), anti-iPLA2 (D-4) (mouse, Santa Cruz Biotechnology, Dallas, TX, USA), anti-HO-1 (rabbit, Proteintech Group, Inc, Rosemont, IL, USA), anti-Nrf2 (rabbit, Abcam, Waltham, MA, USA), anti-GFAP (rabbit, Thermo Fisher Scientific, Waltham, MA, USA), anti-GAPDH (rabbit, Cell Signaling Technology, USA) overnight at 4 °C, followed by horseradish peroxidase (HRP)-linked secondary antibodies (Cell Signaling Technology, Boston, MA, USA) for 1 h at room temperature.

Techniques: Western Blot, Transformation Assay, Two Tailed Test

Journal: Cell

Article Title: Metabolic Fingerprinting Links Oncogenic PIK3CA with Enhanced Arachidonic Acid-Derived Eicosanoids

doi: 10.1016/j.cell.2020.05.053

Figure Lengend Snippet: Oncogenic PIK3CA Signaling Triggers cPLA2-Induced Arachidonic Acid Production (A) Enzymatic activity of cPLA2, iPLA2, and sPLA2 in the MCF10A PIK3CA isogenic panel. (B–D) cPLA2 activity (B) and AA levels (C and D) measured by REIMS in MCF10A H1047R PIK3CA MUT cells following RAPTOR or RICTOR siRNA-mediated knockdown (C), or treatment with 100 nM ASB14780, 1 μM each of PKCα, β, ε, or ζ peptide inhibitors, 250 μM GSK650394, or 150 nM MK2206 for 72 h (D). Cells were grown under exogenous FAF conditions. (E) cPLA2 activity following PKCζ inhibition with 1 μM peptide inhibitor for 72 h. (F and G) Immunoblot analysis of the MCF10A PIK3CA isogenic panel following growth factor deprivation for 16 h and 30 min stimulation with serum and growth factors (F) or PKCζ inhibition with 1 μM peptide inhibitor for 72 h (G). (H) Immunoblot analysis of activated Rac-1 and p38 MAPK in the MCF10A PIK3CA isogenic panel following PKCζ inhibition with 1 μM peptide inhibitor for 72 h. (I) In vitro kinase assay of 100 ng and 0.5 μg/μL purified PKCζ and cPLA2 proteins, respectively. (J) Immunoblot analysis of anti-HA immunoprecipitates derived from HA-tagged cPLA2 transfected MCF10A PIK3CA WT and MUT cells. (K) Immunoblot analysis of anti-HA immunoprecipitates derived from HA-tagged cPLA2 transfected MCF10A PIK3CA WT and MUT cells treated where indicated with 1 μM PKCζ peptide inhibitor for 48 h. (L) AA levels across H1047R MUT cells with CRISPR knockout of PLA2G4A reconstituted with WT or phosphoresistant cPLA2 isoforms. (M) Diagram summarizing the proposed model for PI3K-mTORC2-PKCζ and calcium-dependent activation of cPLA2, leading to a concomitant increase in AA and downstream eicosanoids. Data are presented as the mean ± SEM of n = 3–6 biological replicates and are representative of at least two independent experiments. n.s., not significant; ∗ p ≤ 0.05; ∗∗ p ≤ 0.01; ∗∗∗ p ≤ 0.001. p values in (A) were calculated with unpaired, two tailed Student’s t test, and in (B)–(E), (I), and (L) with one-way ANOVA, followed by unpaired, two-tailed Student’s t test with Bonferroni correction.

Article Snippet: Rabbit polyclonal anti-phospholipase A2 (iPLA2) , Sigma-Aldrich , Cat# SAB4200129; RRID: AB_11129638.

Techniques: Activity Assay, Inhibition, Western Blot, In Vitro, Kinase Assay, Purification, Derivative Assay, Transfection, CRISPR, Knock-Out, Activation Assay, Two Tailed Test

Journal: Cell

Article Title: Metabolic Fingerprinting Links Oncogenic PIK3CA with Enhanced Arachidonic Acid-Derived Eicosanoids

doi: 10.1016/j.cell.2020.05.053

Figure Lengend Snippet: Related to Figure 4 (A) Immunoblot of phospholipases in MCF10A PIK3CA WT and MUT cells following serum and growth-factor deprivation for 16 hours and stimulation with serum and growth factors for 30 min. (B) Immunoblot analysis of cPLA2 protein decay following treatment with 50 μM cycloheximide (CHX) for the indicated times. Image and quantification is from one experiment. (C) Real-time quantitative PCR of PLA2G4A expression in the MCF10A PIK3CA isogenic panel. (D) AA levels measured by REIMS in MCF10A E545K PIK3CA MUT cells following RAPTOR and RICTOR siRNA-mediated knockdown 48 hours post transfection under exogenous FAF conditions. ELISA analysis of (E) AA and (F) PGE2 in the MCF10A PIK3CA isogenic panel 48 hours post RICTOR siRNA-mediated knockdown. (G) Immunoblot analysis of cPLA2 protein decay (top) and quantification (bottom) following RICTOR siRNA-mediated knockdown and treatment with 50 μM cycloheximide for the indicated times. Image and quantification is from one experiment. (H) Immunoblot analysis of substrates of conventional PKCα/β (p-IκBα Ser32 and p-RB Thr821/826), novel PKCε (p-STAT3 Ser727 and p-PKD Ser744/748) and atypical PKCζ (p-RKIP Ser153 and p-cPLA2 T376) isoforms following treatment of MCF10A E545K and H1047R MUT cells with 1 μM of each PKCα, β, ε, and ζ peptide inhibitors for 72 hours. (I) Enzymatic activity of cPLA2, iPLA2, and sPLA2 in the MCF10A PIK3CA isogenic panel following treatment with 100 nM ASB14780 for 72 hours. (J) AA levels measured by REIMS in MCF10A E545K MUT cells treated with 100 nM ASB14780, 1 μM each of PKCα, β, ε, and ζ peptide inhibitors, 250 μM GSK650394, or 150 nM MK2206 for 72 hours under exogenous FAF conditions. (K) Immunoblot (right) of total PKCζ and phospho-S505 and T376 cPLA2 of MCF10A PIK3CA WT and MUT cells following PKCζ siRNA-mediated knockdown, and cPLA2 activity (left) following 48 hours post-transfection. (L) Representative phospho-PKCζ Thr560 immunoreactivity images (left) of 9 PIK3CA MUT (blue) and 9 WT (red) breast PDX tumors. Scale bar = 250 μm. Quantification of percent positive regions (right) was performed using the IHC profiler plug-in for ImageJ. Data are presented as the mean ± SEM of n = 3-5 biological replicates and are representative of at least two independent experiments. n.s., not significant, ∗ p ≤ 0.05; ∗∗ p ≤ 0.01. P value s in (C), (D), (E), (F), (I), (J) and (K, right) with one-way ANOVA followed by unpaired, two-tailed Student’s t test with Bonferroni correction, and in (L) with unpaired, two-tailed Student’s t test.

Article Snippet: Rabbit polyclonal anti-phospholipase A2 (iPLA2) , Sigma-Aldrich , Cat# SAB4200129; RRID: AB_11129638.

Techniques: Western Blot, Real-time Polymerase Chain Reaction, Expressing, Transfection, Enzyme-linked Immunosorbent Assay, Activity Assay, Two Tailed Test

Journal: Cell

Article Title: Metabolic Fingerprinting Links Oncogenic PIK3CA with Enhanced Arachidonic Acid-Derived Eicosanoids

doi: 10.1016/j.cell.2020.05.053

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-phospholipase A2 (iPLA2) , Sigma-Aldrich , Cat# SAB4200129; RRID: AB_11129638.

Techniques: Produced, Recombinant, Transfection, Protease Inhibitor, Lysis, Mutagenesis, Proliferation Assay, In Situ, Calcium Assay, Kinase Assay, Enzyme-linked Immunosorbent Assay, Bicinchoninic Acid Protein Assay, Proximity Ligation Assay, CRISPR, shRNA, Amplification, Plasmid Preparation, Positive Control, Software, Modification, Mass Spectrometry, Western Blot