Journal: Cancers

Article Title: Cathepsin Inhibition Modulates Metabolism and Polarization of Tumor-Associated Macrophages

doi: 10.3390/cancers12092579

Figure Lengend Snippet: ( A ) Detection of prostaglandin E 2 (PGE 2 ) in the medium supernatant of human M0, M1, and M2 macrophages after inhibitor treatment using an enzyme-linked immunosorbent assay (ELISA). ( B ) Expression of mRNA levels of the PGE 2 synthesis enzymes cPLA2 , COX-2, and PGES2 by real-time PCR in M0, M1, and M2 macrophages treated with GB111-NH 2 or DMSO. Data are presented as mean ± standard deviations (SD) and p -values below 0.05 were considered to be statistically significant (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001). ( C ) Representative immunoblot showing cytosolic phospholipase A2 (cPLA2) protein in cells treated as in A. Data are representative for at least three independent experiments, and β-actin served as loading control.

Article Snippet: For immunoblots the following antibodies were used: actin beta (Sigma-Aldrich, Darmstadt, Germany) 1:10,000; acid ceramidase (ASAH1), (Santa Cruz, Heidelberg, Germany) 1:500; autophagy related 16 like 1 (ATG16L1), (Cell Signaling, Frankfurt am Main, Germany) 1:1000; caspase-3 (incl. cleaved form) (Cell Signaling, Frankfurt am Main, Germany) 1:1000; cytosolic phospholipase A2 (cPLA2), (Santa Cruz, Heidelberg, Germany) 1:500; cathepsin B (CTSB), (Santa Cruz, Heidelberg, Germany) 1:1000; cathepsin D (CTSD) (Santa Cruz, Heidelberg, Germany) 1:1.000; cathepsin L (CTSL) (Abcam, Berlin, Germany) 1:500; cathepsin S (CTSS) (Bioss, Boston, MA, USA) 1:500 fatty acid synthase (FASN), (cell signaling, Frankfurt am Main, Germany ) 1:1000; lysosomal associated membrane protein 1 (LAMP-1), (cell signaling, Frankfurt am Main, Germany) 1:1000; microtubule associated protein 1 light chain 3 beta (LC3B), (cell signaling, Frankfurt am Main, Germany) 1:1000; lysosomal phospholipase A2 (lPLA 2 ), (Santa Cruz, Heidelberg, Germany) 1:500; nuclear factor kappa B (NF k Bp65), (cell signaling, Frankfurt am Main, Germany) 1:1000; pNF k Bp65 (cell signaling, Frankfurt am Main, Germany) 1:1000; nitric oxide synthase 2 (NOS2), (Abcam, Berlin, Germany) 1:350; poly(ADP-ribose) polymerase 1 (PARP-1), (cell signaling, Frankfurt am Main, Germany) 1:1000; phosphoinositide kinase, FYVE-type zinc finger containing (PIKFYVE), (Santa Cruz, Heidelberg, Germany) 1:500; transcription factor EB (TFEB), (cell signaling, Frankfurt am Main, Germany) 1:1000; ATPase H+ transporting V1 subunit B1 (V-ATPase B1/2), (Santa Cruz, Heidelberg, Germany) 1:500.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Control

Journal:

Article Title: Differing roles for members of the phospholipase A 2 superfamily in experimental autoimmune encephalomyelitis

doi: 10.1093/brain/awp002

Figure Lengend Snippet: Mean ± SD of the fatty acid hydrolysis

Article Snippet: These cells were then stained with the following antibodies: Polyclonal rabbit anti-cPLA 2 , anti-iPLA 2 , anti-sPLA 2 IIA and anti-sPLA 2 V; monoclonal, FITC-conjugated anti-CD4, anti-CD8, anti-Mac-1/CD11b and anti-CD11c, (BD Pharmingen, 1:200).

Techniques:

Journal: JCI Insight

Article Title: Systems biology identifies cytosolic PLA2 as a target in vascular calcification treatment

doi: 10.1172/jci.insight.125638

Figure Lengend Snippet: (A) Representative cPLA2 and GAPDH Western blots in iliac arteries from early- (n = 3) versus advanced-stage (n = 3) CVD patients. (B) Scatter dot plots (arithmetic mean ± SEM) of cPLA2 protein expression normalized to GAPDH in iliac arteries from early- (n = 14) versus advanced-stage (n = 23) CVD patients. *P < 0.05 vs. early-stage CVD group (unpaired Mann-Whitney test).

Article Snippet: EFEMP1, GUCY1B2, KDM5D, cPLA2, and SHMT1 were detected with a rabbit anti-human EFEMP1 antibody (1:1000, Abcam), rabbit anti-human GUCY1B2 antibody (1:500, Thermo Fisher Scientific), rabbit anti-human KDM5D (Jarid 1D, 1:5000, Novus Biologicals), rabbit anti-human PLA2G4A antibody (1:500, ProSci Incorporated), and rabbit anti-human SHMT1 (1 250, MilliporeSigma), respectively, followed by a goat anti-rabbit IR dye 680 secondary antibody (1:7500, LI-COR Biosciences).

Techniques: Western Blot, Expressing, MANN-WHITNEY

Journal: JCI Insight

Article Title: Systems biology identifies cytosolic PLA2 as a target in vascular calcification treatment

doi: 10.1172/jci.insight.125638

Figure Lengend Snippet: (A) Scatter dot plots (arithmetic mean ± SEM) (n = 5–6, μg/mg protein) of calcium content in aortic tissue of mice receiving vehicle (CTR) or high-dosed cholecalciferol (vD), without or with additional AACOCF3 (AAC) treatment. (B) Scatter dot plots (arithmetic mean ± SEM) (n = 5–6; arbitrary units [a.u.]) of Pla2g4a relative mRNA expression in aortic tissue of mice receiving vehicle or high-dosed cholecalciferol, without or with additional AACOCF3 treatment. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control mice; †P < 0.05 vs. vD-treated mice (1-way ANOVA with Tukey-HSD post-hoc test).

Article Snippet: EFEMP1, GUCY1B2, KDM5D, cPLA2, and SHMT1 were detected with a rabbit anti-human EFEMP1 antibody (1:1000, Abcam), rabbit anti-human GUCY1B2 antibody (1:500, Thermo Fisher Scientific), rabbit anti-human KDM5D (Jarid 1D, 1:5000, Novus Biologicals), rabbit anti-human PLA2G4A antibody (1:500, ProSci Incorporated), and rabbit anti-human SHMT1 (1 250, MilliporeSigma), respectively, followed by a goat anti-rabbit IR dye 680 secondary antibody (1:7500, LI-COR Biosciences).

Techniques: Expressing, Control

Journal: JCI Insight

Article Title: Systems biology identifies cytosolic PLA2 as a target in vascular calcification treatment

doi: 10.1172/jci.insight.125638

Figure Lengend Snippet: Scatter dot plots (arithmetic mean ± SEM) (n = 6, arbitrary units [a.u.]) of PLA2G4A (A), MSX2 (B), CBFA1 (C), and ALPL (D) relative mRNA expression in HAoSMCs following silencing with negative control siRNA (Neg.si) or cPLA2 siRNA (cPLA2si), without or with additional treatment with β-glycerophosphate (Pi). *P < 0.05, **P < 0.01, ***P < 0.001 vs. Neg.si-silenced HAoSMCs; †P < 0.05, †††P < 0.001 vs. Neg.si-silenced and Pi-treated HAoSMCs (1-way ANOVA with Games-Howell post-hoc test for A or with Tukey-HSD post-hoc test for C and Steel-Dwass method for B and D).

Article Snippet: EFEMP1, GUCY1B2, KDM5D, cPLA2, and SHMT1 were detected with a rabbit anti-human EFEMP1 antibody (1:1000, Abcam), rabbit anti-human GUCY1B2 antibody (1:500, Thermo Fisher Scientific), rabbit anti-human KDM5D (Jarid 1D, 1:5000, Novus Biologicals), rabbit anti-human PLA2G4A antibody (1:500, ProSci Incorporated), and rabbit anti-human SHMT1 (1 250, MilliporeSigma), respectively, followed by a goat anti-rabbit IR dye 680 secondary antibody (1:7500, LI-COR Biosciences).

Techniques: Expressing, Negative Control

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: Antioxidants

Article Title: PCSK9 Regulates Nox2-Mediated Platelet Activation via CD36 Receptor in Patients with Atrial Fibrillation

doi: 10.3390/antiox9040296

Figure Lengend Snippet: Intra-signalling pathway of platelet activation PCSK9-mediated: p38MAP Kinase, p47 phox and PLA 2 phosphorylation. p38 phosphorylation, p47 phox phosphorylation and cPLA2 phosphorylation was analysed in platelets incubated with PCSK9 (1–2 ng/mL) and stimulated with subthreshold concentration (STC) of collagen (0.25 µg/mL) ( A–C ) in presence or less of anti-CD36, anti-LOX1, anti-PCSK9 ( A–C ) and incubated with or without SB ( A ), Nox2ds-tat ( B ) and AACOCF3 ( C ) ( n = 3 independent experiments; * p < 0.05 ** p < 0.001 in quantitative analysis). A representative Western blot of p38, p47phox and cPLA2 phosphorylation ( B–C ).

Article Snippet: Moreover, membranes were incubated overnight at 4 °C with rabbit polyclonal anti-p-p47phox (1:1000; ab-795, Abcam, Cambridge, UK), anti-p-p38 and anti-p-cPLA2 antibody (1:1000; sc-7973 and sc34392, Santa Cruz Biotechnology, Dallas, TX, USA; respectively), and anti-p47phox, anti-p38 and anti-cPLA2 antibody (1:1000; sc-17845, sc-7973 and sc-376618, Santa Cruz Biotechnology, TX, USA; respectively).

Techniques: Activation Assay, Phospho-proteomics, Incubation, Concentration Assay, Western Blot