vu0285655-1  (Avanti Polar)

Journal: International Journal of Molecular Sciences

Article Title: Targeting Phospholipase D Pharmacologically Prevents Phagocytic Function Loss of Retinal Pigment Epithelium Cells Exposed to High Glucose Levels

doi: 10.3390/ijms231911823

Figure Lengend Snippet: Non-specific phagocytosis in ARPE-19 cells exposed to inflammatory injury. ( A ) ARPE-19 cells were seeded in 96-well plates and exposed to high glucose concentration (HG, 33 mM) for 24, 48, or 72 h. ( B ) ARPE-19 cells were exposed to vehicle 0.025% DMSO (control condition), PLD1i (0.5 or 5 μM) or PLD2i (0.5 or 5 μM) for 30 min prior to cell exposure to 33 mM HG or to NG (control condition). PLD inhibitors were present during HG treatment. ( C ) ARPE-19 cells were exposed to vehicle 0.025% DMSO (control condition), PLD1i (5 μM), or PLD2i (5 μM) for 48 h in media containing normal glucose concentration (Control condition or NG, 5.5 mM). For A-C, non-specific phagocytosis was measured using pHrodo™ green bioparticles ® conjugates as described in the Materials and Methods section. Representative fluorescence images (Scale bar = 10 µm) are shown; bar graphs show pHrodo fluorescence intensity expressed as arbitrary units (AU) with respect to control conditions. Significant differences with respect to each control condition are indicated with asterisks (*** p < 0.001; ** p < 0.01). ( D ) ARPE-19 cells were exposed to HG (33 mM) or NG (5.5 mM) for 24 h. qPCR assays for the quantification of PLD1 and PLD2 mRNA levels were performed as described in the Materials and Methods section. Results are expressed as 2 -ΔΔCt relative fold mRNA accumulation using GAPDH as internal reference gene. Significant differences with respect to NG are indicated with asterisks (** p < 0.01).

Article Snippet: VU0359595 (PLD1 inhibitor) and VU0285655-1 (PLD2 inhibitor) were from Avanti Polar Lipids, Inc. (Alabaster, AL, USA).

Techniques: Concentration Assay, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Targeting Phospholipase D Pharmacologically Prevents Phagocytic Function Loss of Retinal Pigment Epithelium Cells Exposed to High Glucose Levels

doi: 10.3390/ijms231911823

Figure Lengend Snippet: PLD1 and PLD2 expression and silencing in ABC cells. Confluent ABC cell cultures were transiently transfected for 48 h with PLD1 siRNA, PLD2 siRNA, or PLD1 plus PLD2 siRNAs, as described in the Materials and Methods section. WB assays were performed to detect PLD1 ( A ), PLD2 ( B ), and GAPDH. For A and B, bar graphs show the densitometry values of PLD1/GAPDH and PLD2/GAPDH expressed as arbitrary units (AU) with respect to control conditions (neg control siRNA). ( C ) Heatmap from RNAseq analysis of ARPE-19 cells and hRPE49 cells compared to ABC showing PLD1 and PLD2 expression. Analysis was performed using ABC cells as control (fold change). Red means the pathway is upregulated when compared to ABC, while blue means it is downregulated. Data are detailed in . ( D ) Effect of PLD silencing on mTOR pathway activation. WB was performed to detect activation (phosphorylation) of p70 S6K (p p70 S6K), total p70 S6K, and GAPDH. The bar graph shows the densitometry values of p p70 S6K/p70 S6K expressed as arbitrary units (AU) with respect to control conditions (neg control siRNA). ( E ) Effect of PLD silencing on POS phagocytosis of ABC cells. After siRNA transfection, the medium was removed and ABC cells were incubated with unlabeled POS suspension in MEM at a 1:10 (cell:POS) ratio for 16 h as described in the Materials and Methods section. Total POS (bound and internalized) and internal POS were determined by WB. Bar graphs show the densitometry values of rhodopsin/GAPDH expressed as arbitrary units (AU) with respect to control conditions (neg control siRNA). For ( A , B , D , E ), numbers to the right indicate molecular weights, asterisks (*) indicate significant differences with respect to control condition (*** p < 0.001; ** p < 0.01; * p < 0.05), and whole membranes are depicted in .

Article Snippet: VU0359595 (PLD1 inhibitor) and VU0285655-1 (PLD2 inhibitor) were from Avanti Polar Lipids, Inc. (Alabaster, AL, USA).

Techniques: Expressing, Transfection, Activation Assay, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Targeting Phospholipase D Pharmacologically Prevents Phagocytic Function Loss of Retinal Pigment Epithelium Cells Exposed to High Glucose Levels

doi: 10.3390/ijms231911823

Figure Lengend Snippet: Schematic view of PLD-mediated events elicited in RPE cells exposed to inflammatory injury. ( A ) The PLD pathway is activated in RPE cells exposed to inflammatory injury to mediate reactive oxygen species (ROS) generation and nuclear factor kappa B (NFκB)-mediated inflammatory response, leading to a reduced RPE cell viability and phagocytic function. ( B ) Pharmacological inhibition of PLD1 and PLD2 is able to prevent ROS generation and the RPE inflammatory response, resulting in a normal phagocytic function. Dashed arrows indicate possible but not fully elucidated mechanisms.

Article Snippet: VU0359595 (PLD1 inhibitor) and VU0285655-1 (PLD2 inhibitor) were from Avanti Polar Lipids, Inc. (Alabaster, AL, USA).

Techniques: Inhibition

Journal: International Journal of Molecular Sciences

Article Title: Targeting Phospholipase D Pharmacologically Prevents Phagocytic Function Loss of Retinal Pigment Epithelium Cells Exposed to High Glucose Levels

doi: 10.3390/ijms231911823

Figure Lengend Snippet: Primer sequences for the specific amplification of human PLD1, PLD2 , and GAPDH .

Article Snippet: VU0359595 (PLD1 inhibitor) and VU0285655-1 (PLD2 inhibitor) were from Avanti Polar Lipids, Inc. (Alabaster, AL, USA).

Techniques: Amplification, Sequencing

Journal: The New Phytologist

Article Title: Arabidopsis phospholipid modifications mediate cellulase‐induced resistance to a fungal peptide antibiotic by imposing cell polarity

doi: 10.1111/nph.70721

Figure Lengend Snippet: PHOSPHOLIPASE Dζ inhibitors prevent cellulase‐induced resistance to alamethicin (CIRA). (a) The effect of the inhibitors VU0359595 (VU1) and VU0285655‐1 (VU2) on CIRA was tested in wild‐type (WT), cira12 and cira13 mutants. WT, cira12 and cira13 mutants were treated overnight with the inhibitors followed by cellulase treatment and then tested for alamethicin‐dependent ion release. A significant increase in alamethicin‐dependent ion release was observed in cira13 mutants post‐VU2 and VU1 + VU2 treatment. Seedlings pretreated with cellulase followed by alamethicin were used as controls. Data were normalised by dividing by the mean of the WT control, the average of which was 0.57 ± 0.02 μS cm −1 per seedling (mean ± SE). Values marked by the same letter are not statistically different ( P < 0.05). Error bars show SE of the mean ( n = 3). Statistical analyses were carried out using one‐way analysis of variance (ANOVA) followed by Tukey post‐hoc analysis (denoted by letters). (b, c) External application of PLDζ‐ inhibitors (VUs) prevents cellulase‐induced anticlinal asymmetry in the plasma membrane (PM) of the WT. (b) Root early extension zone epidermis of Arabidopsis WT and mutant seedlings cira12‐3 expressing the distribution probe mCIT‐tagged phosphatidylserine (PS) sensor (mCIT‐C2 LACT ) treated with and without cellulase and/or with and without PLDζ‐ inhibitors were analysed by spinning disc confocal microscopy. Magenta arrows highlight dual anticlinal PMs separated by a cell wall; white arrows indicate the outer periclinal PM. Bar, 10 μm. (c) Quantified PS sensor fluorescence intensity ratio between outer periclinal and anticlinal PM domains in root epidermal cells with and without cellulase treatment and/or with and without PLDζ‐ inhibitors. Asterisks denote significant difference (***, P < 0.001) in the ratio of fluorescence intensity between mCIT‐C2 LACT /WT and mCIT‐C2 LACT /cira12‐3 . Ratios have been corrected for the anticlinal signal coming from two membranes in close proximity. Error bars show SE of the mean ( n = 30). Statistical analyses were carried out using Student’s t ‐test (denoted by asterisks).

Article Snippet: Seedlings grown as mentioned previously were washed with water and treated overnight with 300 nM PLDζ inhibitors VU1 (VU0359595 Avanti Polar Lipids), and/or VU2 (VU0285655‐1 Avanti Polar Lipids) in 1⁄2 MS (procedure modified from Yao et al ., ) (Yao et al ., ).

Techniques: Control, Clinical Proteomics, Membrane, Mutagenesis, Expressing, Confocal Microscopy, Fluorescence

Journal: Autophagy

Article Title: Phosphatidic acid suppresses autophagy through competitive inhibition by binding GAPC (glyceraldehyde-3-phosphate dehydrogenase) and PGK (phosphoglycerate kinase) proteins.

doi: 10.1080/15548627.2022.2046449

Figure Lengend Snippet: Figure 1. Phosphatidic acid (PA) suppresses autophagosome formation in Arabidopsis roots. (A) GFP-ATG8e–labeled dots and ring-like structures in elongation zone (EZ) of Arabidopsis roots after 1-butanol treatment. Arabidopsis seedlings expressing GFP-ATG8e fusion protein were grown on 1/2 MS medium for 5 days, then transferred to 1/2 MS medium containing 1-butanol (0.4%) for 12 h in the presence of concanamycin A (Con A, 0.5 μM) and visualized by confocal microscopy. Representative images were shown (upper, bar: 50 μm). The GFP-ATG8e dots or ring-like structures are indicated by arrows and enlarged (bottom, bar: 5 μm). (B) Observation (left, bars = 50 μm) and measurement (right) of puncta per root section of GFP-ATG8e–labeled dots and ring-like structures in the meristem zone (MZ) and EZ of Arabidopsis roots under treatment with phosphatidic acid (PA), 1-butanol or PLDζ inhibitor. Arabidopsis seedlings expressing GFP-ATG8e fusion protein were grown on 1/2 MS medium for 5 days, then transferred to 1/2 MS medium containing PA (10 μM), 1-butanol (0.4%) or PLDζ inhibitor (200 nM) for 12 h in the presence of Con A (0.5 μM) and visualized by confocal microscopy. Representative images were shown. Experiments were biologically repeated for three times and data were shown as mean ± SD (n = 15). Statistical significance was determined by Student's t-test (*, P < 0.05; **, P < 0.01; ***, P < 0.001, compared with control). (C) Immunodetection of free GFP release during autophagy-mediated vacuolar degradation of GFP-ATG8e under PA, 1-butanol or PLDζ inhibitor treatment. Arabidopsis seedlings expressing GFP-ATG8e fusion protein were grown on 1/2 MS medium for 7 days, then transferred to 1/2 MS medium containing PA (10 μM), 1-butanol (0.4%) or PLDζ inhibitor (200 nM) for 12 h in the presence of Con A (0.5 μM), followed by protein extraction and immunoblotting analyses. The experiments were biologically repeated for three times and ACTIN was used as a protein loading control.

Article Snippet: To determine the effect of PA on autophagosome formation, 5-day-old seedlings expressing eGFP-ATG8e grown on 1/2 MS medium (PhytoTechnology Laboratories, M519) were transferred to 1/2 MS medium containing PA (10 μM; Avanti Polar Lipids, 840875), 1-butanol (0.4%; Sigma, 281549), or PLDζ inhibitor (200 nM; Avanti Polar Lipids, VU0285655-1), in the absence or presence of concanamycin A (Con A, 0.5 μM; Sangon Biotech, A601179) and grown under normal conditions.

Techniques: Labeling, Expressing, Confocal Microscopy, Control, Immunodetection, Protein Extraction, Western Blot