Journal: bioRxiv

Article Title: DLGAP1-AS2-Mediated Phosphatidic Acid Synthesis Confers Chemoresistance via Activation of YAP Signaling

doi: 10.1101/2022.02.24.481869

Figure Lengend Snippet: ( A ) IB detection of YAP and LATS1 phosphorylation. KYSE450 cells expressing empty vector (EV) and D-AS2 were pretreated with FIPI (30 µM) or CAY10594 (20 µM) for 1 h. ( B ) RT-qPCR detection of the YAP target genes CTGF and CYR61. KYSE450 cells expressing EV and D-AS2 were pretreated with FIPI (30 µM) or CAY10594 (20 µM) for 1 h. The data are presented as the mean ± s.d. values; two-tailed t test, *** P < 0.001; n = 3. ( C, D ) IF detection of YAP localization in KYSE450 cells expressing EV and D-AS2. Cells were pretreated with FIPI (30 µM) or CAY10594 (20 µM). YAP localization is shown in (C); scale bar, 30 µm. Cells from three different fields in three independent experiments were randomly selected for quantification of YAP localization (D). The data are expressed as the mean ± s.e.m. values. ( E ) IB detection of YAP and LATS1 phosphorylation. Serum-starved KYSE30 cells expressing control or D-AS2-targeting sgRNAs were pretreated with PA (100 µM) for 1 h. ( F ) RT-qPCR detection of the YAP target genes CTGF and CYR61. Serum-starved KYSE30 cells expressing control or D-AS2-targeting sgRNAs were pretreated with PA (100 µM) for 1 h. The data are presented as the mean ± s.d. values; two-tailed t test, *** P < 0.001, ns: not significant; n = 3. ( G, H ) IF detection of YAP localization in KYSE30 cells expressing control and D-AS2-targeting sgRNAs and pretreated with PA (100 µM) for 1 h. YAP localization is shown in (G); scale bar, 30 µm. Cells from three different fields in three independent experiments were randomly selected for quantification of YAP localization (H). The data are expressed as the mean ± s.e.m. values. ( I ) RT-qPCR detection of the indicated genes expression in the samples with chemoresistance or chemosensitivity of esophageal SCC patients. The data are presented as the mean ± s.e.m. values; two-tailed t test; n = 17 for chemoresistant patients; n = 17 for chemosensitive patients.

Article Snippet: FIPI (939055-18-2) and CAY10594 (1130067-34-3) were purchased from Topscience (Topscience; Shanghai, China).

Techniques: Expressing, Plasmid Preparation, Quantitative RT-PCR, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: A new model for regulation of sphingosine kinase 1 translocation to the plasma membrane in breast cancer cells

doi: 10.1016/j.jbc.2021.100674

Figure Lengend Snippet: Effect of the PLD inhibitor, FIPI, or the G q inhibitor YM254890 on the translocation of WT GFP- m SK1 in MCF-7L cells. MCF-7L cells overexpressing WT GFP- m SK1 were pretreated with and without FIPI (100 nM, 1 h) or YM254890 (10 μM, 30 min) before S1P (5 μM) or PMA (1 μM) or carbachol (100 μM) for 10 min. Cells were processed and mounted with DAPI to stain the DNA ( blue ). A , photomicrographs of cells of 40× oil magnification overexpressing WT GFP- m SK1 detected with GFP. Representative results of three independent experiments. B , the bar graphs ( lower of each pair ) represent the AUC of transfected WT GFP- m SK1 translocation (n = 5) and ( upper of each pair ) the percentage of cells containing translocated WT GFP- m SK1 (n = 3); ∗ p < 0.05, ∗ ∗ p < 0.01, ∗ ∗ ∗ p < 0.001, and ∗ ∗ ∗ ∗ p < 0.0001 for stimulus alone versus stimulus with either FIPI or YM254890; + p < 0.05, ++ p < 0.01, +++ p < 0.001, and ++++ p < 0.0001 for stimulus versus control transfected WT GFP- m SK1 (two-way ANOVA with Tukey's post hoc test). AUC, area under the curve; FIPI, N -[2-[4-(2,3-dihydro-2-oxo-1 H -benzimidazol-1-yl)-1-piperidinyl]ethyl]-5-fluoro-1 H -indole-2-carboxamide hydrochloride; m SK1, mouse SK1; PLD, phospholipase D; PMA, phorbol 12-myristate 13-acetate; S1P, sphingosine-1-phosphate; SK1, sphingosine kinase 1.

Article Snippet: S1P was from Avanti Polar Lipids, YM254890 from Caltag Medsystems, FIPI from Cayman Chemical Company, and PD98059 from Sigma-Aldrich Company Ltd. Antibodies used were anti-GAPDH (sc-47724), anti-GFP (sc-9996), and anti-phosphoERK1/2 (sc-7383) from Santa Cruz (through Insight Biotechnology Ltd), anti-ERK2 (#610104) from BD Biosciences, and anti-HA tag antibody (H3663) and anti-actin (A2066) were from Sigma-Aldrich Company Ltd. Anti-fascin (#54545), anti-paxillin (#2542), and anti-cortactin (#3503) antibodies were purchased from Cell Signaling Technology.

Techniques: Translocation Assay, Staining, Transfection

Journal: Pharmacological Reviews

Article Title: Phospholipase D Signaling Pathways and Phosphatidic Acid as Therapeutic Targets in Cancer

doi: 10.1124/pr.114.009217

Figure Lengend Snippet: Modulators of PLD activity

Article Snippet: However, there was no mention of PLD1 inhibition by the Novartis group, but it was subsequently found that halopemide (compound 7) potently inhibits both PLD1 (cellular IC 50 = 21 nM, biochemical IC 50 = 220 nM) and PLD2 (cellular IC 50 = 300 nM, biochemical IC 50 = 310 nM) as does FIPI 7 (PLD1 cellular IC 50 = 1 nM, biochemical IC 50 = 9.5 nM; PLD2 cellular IC 50 = 44 nM, biochemical IC 50 = 17 nM).

Techniques:

Journal: Pharmacological Reviews

Article Title: Phospholipase D Signaling Pathways and Phosphatidic Acid as Therapeutic Targets in Cancer

doi: 10.1124/pr.114.009217

Figure Lengend Snippet: Structures and cellular PLD activity of halopemide (7) and the related analog FIPI (8), dual PLD1/PLD2 inhibitors with classic atypical antipsychotic promiscuous ancillary pharmacology.

Article Snippet: However, there was no mention of PLD1 inhibition by the Novartis group, but it was subsequently found that halopemide (compound 7) potently inhibits both PLD1 (cellular IC 50 = 21 nM, biochemical IC 50 = 220 nM) and PLD2 (cellular IC 50 = 300 nM, biochemical IC 50 = 310 nM) as does FIPI 7 (PLD1 cellular IC 50 = 1 nM, biochemical IC 50 = 9.5 nM; PLD2 cellular IC 50 = 44 nM, biochemical IC 50 = 17 nM).

Techniques: Activity Assay

Journal: Pharmacological Reviews

Article Title: Phospholipase D Signaling Pathways and Phosphatidic Acid as Therapeutic Targets in Cancer

doi: 10.1124/pr.114.009217

Figure Lengend Snippet: Chemical optimization strategy for halopemide (7) employing a combination of diversity-oriented synthesis, and more focused iterative parallel synthesis. This effort led to the development of the 1700-fold PLD1-preferring inhibitor VU0359595 (9), the 75-fold PLD2-preferring VU0364739 (10), the >50-fold PLD2-selective MLPCN probe ML298 (11), and the potent dual PLD1/PLD2 inhibitor MLPCN probe ML299 (12). Importantly, ancillary pharmacology was improved significantly over compound 7 as well as PLD isoform selectivity.

Article Snippet: However, there was no mention of PLD1 inhibition by the Novartis group, but it was subsequently found that halopemide (compound 7) potently inhibits both PLD1 (cellular IC 50 = 21 nM, biochemical IC 50 = 220 nM) and PLD2 (cellular IC 50 = 300 nM, biochemical IC 50 = 310 nM) as does FIPI 7 (PLD1 cellular IC 50 = 1 nM, biochemical IC 50 = 9.5 nM; PLD2 cellular IC 50 = 44 nM, biochemical IC 50 = 17 nM).

Techniques:

Journal: Frontiers in Physiology

Article Title: Enzymatic Activity Is Not Required for Phospholipase D Mediated TNF-α Regulation and Myocardial Healing

doi: 10.3389/fphys.2018.01698

Figure Lengend Snippet: FIPI has no influence on infarct size or left ventricular function 24 h post MI in mouse hearts. (A) Quantitative analysis of infarct size as the percentage of area at risk (% Inf/AaR), of the area at risk as percentage of the left ventricle and of the infarct size as percentage of the left ventricle showed no differences in comparison of the control and FIPI-treated group in TTC-stained left ventricles 24 h after I/R (control n = 12, FIPI n = 13). (B) Representative pictures. Blue = healthy tissue, red = the area at risk (AAR), white = infarcted area (INF). (C) Echocardiographic analysis of ejection fraction (baseline vs. 24 h after I/R), cardiac output (baseline vs. 24 h after I/R) and fractional shortening (baseline vs. 24 h after I/R) of C57BL6/J mice treated with FIPI (3 mg/kg bodyweight in 4% DMSO/PBS) vs. controls showed no differences (control n = 20, FIPI n = 23).

Article Snippet: For the analysis of the impact of PLD enzymatic activity on myocardial ischemia and reperfusion injury, treatment of C57BL/6J mice (Janvier Labs) with the PLD inhibitor FIPI were performed.

Techniques: Comparison, Control, Staining

Journal: Frontiers in Physiology

Article Title: Enzymatic Activity Is Not Required for Phospholipase D Mediated TNF-α Regulation and Myocardial Healing

doi: 10.3389/fphys.2018.01698

Figure Lengend Snippet: Pharmacological inhibition of PLD enzymatic activity reduces the inflammatory response 24 h post MI in mice. Twenty four hours post MI, cardiac sections of FIPI-treated mice (3 mg/kg bodyweight in 4% DMSO/PBS) vs. controls were stained with either hematoxylin/eosin (A) or immune cell specific antibodies for neutrophils (B) and macrophages (C) to analyze the migration of inflammatory cells into the infarct border zone (n = 5). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. (D,E) Flow cytometric analysis of platelet-immune cell-aggregate formation 0, 24, and 72 h post MI in FIPI-treated mice vs. controls; Platelet – leukocyte-aggregate formation left, platelet – neutrophile – aggregate formation right ( n = 5–21). (F) Quantitative analysis of IL-1β in plasma of FIPI-treated mice vs. control 24 h post MI (control n = 14; FIPI n = 16). (G) Analysis of IL-1β expression in the left ventricle of Pld1 +/+ /Pld2 +/+ vs. Pld1 -/- /Pld2 -/- mice 24 h post MI using quantitative RT-PCR ( Pld1 +/+ /Pld2 +/+ n = 3, Pld1 -/- /Pld2 -/- n = 4). (H) Quantitative analysis of IL-6 expression in the left ventricle of FIPI-treated vs. control mice 24 h post MI (control n = 5; FIPI n = 6). Scale bar: 50 μm, magnification: 400×.

Article Snippet: For the analysis of the impact of PLD enzymatic activity on myocardial ischemia and reperfusion injury, treatment of C57BL/6J mice (Janvier Labs) with the PLD inhibitor FIPI were performed.

Techniques: Inhibition, Activity Assay, Staining, Migration, Clinical Proteomics, Control, Expressing, Quantitative RT-PCR

Journal: Frontiers in Physiology

Article Title: Enzymatic Activity Is Not Required for Phospholipase D Mediated TNF-α Regulation and Myocardial Healing

doi: 10.3389/fphys.2018.01698

Figure Lengend Snippet: Treatment with FIPI shows no alterations in cell apoptosis after I/R. (A) Analysis of quantitative RT-PCR shows no differences in the expression of the cell apoptosis markers Bax, Bcl-xl , and Bcl -2 in the left ventricle (LV) of FIPI-treated mice (3 mg/kg bodyweight in 4% DMSO/PBS) vs. control mice 24 h post MI (control n = 5, FIPI n = 6). (B) Number of caspase-3 positive cells in the infarct border zone is not different between FIPI-treated vs. control mice 21 days post MI (control n = 4, FIPI n = 5) (C) After I/R in vitro the amount of activated caspase3 in HL-1 cells is not altered after FIPI-treatment ( n = 4). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: For the analysis of the impact of PLD enzymatic activity on myocardial ischemia and reperfusion injury, treatment of C57BL/6J mice (Janvier Labs) with the PLD inhibitor FIPI were performed.

Techniques: Quantitative RT-PCR, Expressing, Control, In Vitro

Journal: Frontiers in Physiology

Article Title: Enzymatic Activity Is Not Required for Phospholipase D Mediated TNF-α Regulation and Myocardial Healing

doi: 10.3389/fphys.2018.01698

Figure Lengend Snippet: FIPI treatment has no influence on scar formation 21 d post MI. (A) No alterations in infarct size ( n = 6) and (B) left ventricular function 21 days post MI in FIPI (3 mg/kg bodyweight in 4% DMSO/PBS) treated mice vs. controls ( n = 14–18). (C) Sirius red staining of remote myocardium shows no differences in the amount of interstitial collagen between FIPI-treated mice vs. controls (control n = 9, FIPI n = 6) (D) Quantification of α-SMA positive myofibroblasts in the border zone of infarcted hearts 21 days post MI in FIPI-treated mice vs. control mice shows no alterations between the groups. Representative pictures are shown (control n = 9, FIPI n = 6). Scale bar: 50 μm, magnification: 400×.

Article Snippet: For the analysis of the impact of PLD enzymatic activity on myocardial ischemia and reperfusion injury, treatment of C57BL/6J mice (Janvier Labs) with the PLD inhibitor FIPI were performed.

Techniques: Staining, Control

Journal: Frontiers in Physiology

Article Title: Enzymatic Activity Is Not Required for Phospholipase D Mediated TNF-α Regulation and Myocardial Healing

doi: 10.3389/fphys.2018.01698

Figure Lengend Snippet: Hemodynamic measurements after MI.

Article Snippet: For the analysis of the impact of PLD enzymatic activity on myocardial ischemia and reperfusion injury, treatment of C57BL/6J mice (Janvier Labs) with the PLD inhibitor FIPI were performed.

Techniques: Control

Journal: Frontiers in Physiology

Article Title: Enzymatic Activity Is Not Required for Phospholipase D Mediated TNF-α Regulation and Myocardial Healing

doi: 10.3389/fphys.2018.01698

Figure Lengend Snippet: TNF-α signaling is independent of the enzymatic activity of PLD. (A) TNF-α expression in the left ventricle of infarcted hearts shows no alterations in FIPI-treated (3 mg/kg bodyweight in 4% DMSO/PBS) vs. control mice 24 h post MI (control n = 5, FIPI n = 6). (B) TNF-α expression in the left ventricle of Pld1 +/+ /Pld2 +/+ vs. Pld1 -/- /Pld2 -/- mice 24 h post MI was significantly decreased in mutant mice ( Pld1 +/+ /Pld2 +/+ n = 3/4; Pld1 -/- /Pld2 -/- n = 3/5). (C) Pld1 +/+ -MEFs were treated with FIPI (1 μM in DMSO/PBS, 30 min prior LPS treatment), stimulated with LPS (1 μg/ml) for different time periods as indicated and the TNF-α release was measured via ELISA. No differences in TNF-α release were observed in FIPI-treated MEFs compared to untreated controls ( n = 5). (D) Pld1 -/- - MEFs were treated with FIPI (1 μM in DMSO/PBS) and stimulated with LPS (1 μg/ml) for 12 h. TNF-α release was measured by ELISA. TNF-α release of Pld1 -/- -MEFs and Pld1 -/- -MEFs treated with FIPI was decreased in comparison to Pld1 +/+ - MEFs ( n = 5). LV = left ventricle. ∗∗ P < 0.01 and ∗∗∗ P < 0.001.

Article Snippet: For the analysis of the impact of PLD enzymatic activity on myocardial ischemia and reperfusion injury, treatment of C57BL/6J mice (Janvier Labs) with the PLD inhibitor FIPI were performed.

Techniques: Activity Assay, Expressing, Control, Mutagenesis, Enzyme-linked Immunosorbent Assay, Comparison