Journal: The Journal of Biological Chemistry

Article Title: Phosphatidic Acid Produced by RalA-activated PLD2 Stimulates Caveolae-mediated Endocytosis and Trafficking in Endothelial Cells *

doi: 10.1074/jbc.M116.752485

Figure Lengend Snippet: RalA siRNA and PLD inhibitors block caveolae-mediated endocytosis of BSA. A, starved HLMVECs treated with SMART POOL (SP) RalA siRNA or individually with each of the four oligonucleotides for 48 h were incubated with Alexa-488-BSA for 30 min, acid-washed, and fixed. Images of internalized fluorescent albumin were acquired by confocal microscopy. B, quantification of fluorescence intensity of Alexa-488-BSA demonstrates that RalA siRNA blocked BSA uptake (n = 10/group; ***, p < 0.001 versus siCont by ANOVA). C, HLMVECs treated with SMART POOL RalA siRNA for 48 h were transfected with RalA-GFP to rescue RalA expression as compared with GFP, which was transfected as a negative control. Cells were then lysed and examined by Western blotting (IB) to confirm RalA rescue. D, quantification of fluorescence intensity of Alexa-555-BSA uptake in RalA-depleted and -repleted HLMVECs (n = 10/group; ***, p < 0.001 by ANOVA). E, quantification of fluorescence intensity of Alexa-488-BSA demonstrates that both RalA knockdown by siRNA and 1-butanol dramatically inhibited BSA uptake (n = 6/group; ***, p < 0.001 versus control siRNA by ANOVA). F, HLMVECs treated with 200 nm VU0359595 (PLD1 inhibitor) or VU0364739 (PLD2 inhibitor) for 1 h were incubated with Alexa-488-BSA for 30 min. Quantification of Alexa-488-BSA fluorescence indicates that PLD2 inhibitor but not PLD1 inhibitor blocked albumin uptake (n = 7/group; ***, p < 0.001 versus control by ANOVA). Error bars, S.E.

Article Snippet: PLD1 inhibitor VU0359595 was from Cayman Chemical (Ann Arbor, MI), and PLD2 inhibitor VU 0364739 was from Tocris Bioscience (Bristol, UK).

Techniques: Blocking Assay, Incubation, Confocal Microscopy, Fluorescence, Transfection, Expressing, Negative Control, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Phosphatidic Acid Produced by RalA-activated PLD2 Stimulates Caveolae-mediated Endocytosis and Trafficking in Endothelial Cells *

doi: 10.1074/jbc.M116.752485

Figure Lengend Snippet: Cav-1-RFP-positive vesicle trafficking and fusion. A, Western blotting analysis of overexpressed hPLD1 K898R (PLD1 mutant) and mPLD2 K758R (PLD2 mutant) versus EV and control cells. B, HLMVECs were infected with EV, hPLD1 K898R, or mPLD2 K758R mutant in adenovirus, labeled with [32P]ATP, and stimulated with thrombin (0.005 units/ml). [32P]PBt formation as a result of PLD activation indicated that PLD2 mutant inhibited thrombin-induced PLD activity, whereas PLD1 mutant had no effect. n = 3/group; *, p < 0.05; **, p < 0.01 by ANOVA. C, HLMVECs transfected with Cav-1-RFP and infected with EV, hPLD1-K898R, or mPLD2 K758R mutant were serum-deprived for 2 h, treated with 30 mg/ml BSA, and then imaged by live cell TIRF microscopy every 5 min for 20 min. Note the time-dependent appearance of Cav-1-RFP in the TIRF plane (abluminal aspect of the cell). D, quantification of relative fluorescence intensity of Cav-1-RFP (n = 3 regions/group from three independent experiments; **, p < 0.01 versus control; ***, p < 0.001 versus EV control + BSA by ANOVA). E, HLMVECs transfected with Cav-1-RFP and after 24 h were serum-deprived for 2 h, treated with 200 nm VU0359595 (PLD1 inhibitor) or VU0364739 (PLD2 inhibitor) for 1 h, and then stimulated with 30 mg/ml BSA and imaged by live cell TIRF microscopy every 5 min for 20 min (n = 3 regions/group from three independent experiments; ***, p < 0.001 versus control without BSA; **, p < 0.01 versus control with BSA by ANOVA). Error bars, S.E. AU, arbitrary units.

Article Snippet: PLD1 inhibitor VU0359595 was from Cayman Chemical (Ann Arbor, MI), and PLD2 inhibitor VU 0364739 was from Tocris Bioscience (Bristol, UK).

Techniques: Western Blot, Mutagenesis, Infection, Labeling, Activation Assay, Activity Assay, Transfection, Microscopy, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Phosphatidic Acid Produced by RalA-activated PLD2 Stimulates Caveolae-mediated Endocytosis and Trafficking in Endothelial Cells *

doi: 10.1074/jbc.M116.752485

Figure Lengend Snippet: Effect of PLD2 mutant on PA generation in caveolae. A, HLMVECs were co-transfected with GFP-PASS and Cav-1-RFP following infection with EV, hPLD1-K898R, or mPLD2 K758R mutant; serum-deprived for 2 h; treated with 30 mg/ml BSA for 30 min; and imaged by confocal microscopy. B, confocal images representative of three independent experiments were used to determine Pearson's correlation coefficient of co-localized GFP-PASS and Cav-1-RFP fluorescence. Results indicate that BSA increases and PLD2 mutant reduces co-localization of PA and Cav-1 (n = 10/group; ***, p < 0.001 versus EV + BSA by ANOVA). Error bars, S.E.

Article Snippet: PLD1 inhibitor VU0359595 was from Cayman Chemical (Ann Arbor, MI), and PLD2 inhibitor VU 0364739 was from Tocris Bioscience (Bristol, UK).

Techniques: Mutagenesis, Transfection, Infection, Confocal Microscopy, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Phosphatidic Acid Produced by RalA-activated PLD2 Stimulates Caveolae-mediated Endocytosis and Trafficking in Endothelial Cells *

doi: 10.1074/jbc.M116.752485

Figure Lengend Snippet: Proposed model for role of RalA in caveolae-mediated endocytosis. In the presence of albumin, RalA is recruited to caveolae, where it associates with Cav-1 and FilA, ultimately becoming activated upon GTP binding. Activated RalA triggers downstream effector PLD2-mediated generation of PA, which promotes caveolae-mediated endocytosis and transcytosis.

Article Snippet: PLD1 inhibitor VU0359595 was from Cayman Chemical (Ann Arbor, MI), and PLD2 inhibitor VU 0364739 was from Tocris Bioscience (Bristol, UK).

Techniques: Binding Assay

Journal: BMC biology

Article Title: Phosphatidic acid-dependent localization and basal de-phosphorylation of RA-GEFs regulate lymphocyte trafficking.

doi: 10.1186/s12915-020-00809-0

Figure Lengend Snippet: Fig. 6 PA-dependent Rap1-GTP localization at the plasma membrane induces the development of the front membrane. a (Left) The FRET-based Rap1 activity sensor-expressing BAF cells were stimulated with CXCL12 in the presence or absence of CAY10594. An image of mTurquoise/Venus ratio represents FRET efficiency. (Center) The FRET efficiency at 6 μm edge region of plasma membrane is shown after CXCL12 stimulation (n = 20). Values are normalized to the level at zero point. The blue arrow marks the addition of CXCL12. (Right) The percentages of cells showing more than 1.3-fold increase in the FRET efficiency at the plasma membrane at 120 s after CXCL12 stimulation are shown (n = 20). *P < 0.001 versus CXCL12-stimulated cells without CAY10594. b (Left upper) Co-localization of PASS-GFP (PA) and Ral-GDS-RBD-mCherry (Rap1-GTP) in BAF cells at 10 min after CXCL12 stimulation with or without CAY10594 is shown. (Lower) We measured the ratios of Ral-GDS localized in the cytoplasm, plasma membrane, and PASS-concentrated region of plasma membrane of the cells. The graph shows percentages of cells showing that more than 50% of Ral-GDS was localized in each region (n = 30). (Right upper) Localization of Spa1-GFP and Ral-GDS-RBD-mCherry in BAF cells treated with CXCL12 is shown. (Lower) We measured the ratios of Spa1 localized in the cytoplasm, plasma membrane, and Ral-GDS- concentrated region of plasma membrane of the cells. The graph shows percentages of cells showing that more than 50% of Spa1 was localized in each region (n = 30). c Distribution of the Ral-GDS-RBD-mCherry and FITC-conjugated anti-CD44 in BAF cells on CXCL12 and ICAM-1-coated surface with (lower) or without (upper) CAY10594 is shown. Time 0 represents the first time-lapse image; subsequent images were obtained in the same focal plane. The asterisk symbol (*) shows the certain position. d Localization of PASS-GFP and Ral-GDS-RBD-mCherry in BAF cells on CXCL12 and ICAM-1-coated surface is shown. Each bar graph represents the means ± SEM. Scale bar, 5 μm

Article Snippet: 0.5 μM staurosporine (protein kinase inhibitor) (Wako Pure Chemicals), 0.5 μM okadaic acid (PP2A inhibitor) (Wako), 5–10 μM R59022 (Tocris Bioscience) (DGK inhibitor), 1 μM dasatinib (abl family PTK inhibitor) (Carbosynth), and 1–2 μM CAY10594 (PLD2 inhibitor) or CAY10593 (PLD1 inhibitor) (Cayman Chemical) were used for examination of their roles in chemokinedependent Rap1 regulation.

Techniques: Clinical Proteomics, Membrane, Activity Assay, Expressing

Journal: eLife

Article Title: Ral GTPases promote breast cancer metastasis by controlling biogenesis and organ targeting of exosomes

doi: 10.7554/elife.61539

Figure Lengend Snippet: Figure 2. The RalA/B-PLD1-PA axis governs exosome secretion. (a) Representative confocal images of 4T1 cells co-transfected with PLD1-GFP and tdTomato-RalA (upper panels) or tdTomato-RalB (lower panels) and incubated with Lysotracker. Scale bar: 10 mm; zoom: 2 mm. (b) Electron microscopy analysis of 4T1 cells treated with PLD1 or PLD2 inhibitor. Scale bar: 1 mm. Violin plots show quantification of the number of multi-vesicular body (MVB) per cytoplasmic surface. Each dot represents one field of view; horizontal bar represents the average (180–194 fields of view; Kruskal-Wallis test Figure 2 continued on next page

Article Snippet: Drug treatment Cells were incubated with the following drugs in the appropriate medium: RalA/B inhibitors BQU57 (10 mM; Sigma) and RBC8 (10 mM; Sigma), PLD1 inhibitor CAY10593 (10 mM; Santa Cruz Biotechnol- ogy) or PLD2 inhibitor CAY10594 (10 mM; Santa Cruz Biotechnology).

Techniques: Transfection, Incubation, Electron Microscopy

Journal: Developmental cell

Article Title: Binding of PLD2-generated phosphatidic acid to KIF5B promotes MT1-MMP surface trafficking and lung metastasis of mouse breast cancer cells

doi: 10.1016/j.devcel.2017.09.012

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit Anti-PLD2 (1:500 for WB) , Cell Signaling , Cat# 13904.

Techniques: Super-Resolution Microscopy, Virus, Recombinant, Protease Inhibitor, Western Blot, Blocking Assay, Membrane, Cloning, Labeling, Transgenic Assay, Plasmid Preparation, Mutagenesis, shRNA, Software, Pore Size

Journal: Mediators of Inflammation

Article Title: Blockade of PLD2 Ameliorates Intestinal Mucosal Inflammation of Inflammatory Bowel Disease

doi: 10.1155/2016/2543070

Figure Lengend Snippet: The primers using in qRT-PCR analysis.

Article Snippet: One group of mice was administered with PLD2 selective inhibitor (CAY10694, Santa Cruz Biotechnology, 4 mg/kg) daily by oral gavage, and another group of mice were administered with PBS as controls.

Techniques: Sequencing

Journal: Mediators of Inflammation

Article Title: Blockade of PLD2 Ameliorates Intestinal Mucosal Inflammation of Inflammatory Bowel Disease

doi: 10.1155/2016/2543070

Figure Lengend Snippet: PLD2 is highly expressed in patients with active IBD. (a) Peripheral blood samples were collected from patients with active CD (A-CD, n = 25), patients with CD in remission (R-CD, n = 19), patients with active UC (A-UC, n = 20), patients with UC in remission (R-UC, n = 21), and healthy controls ( n = 28). Expression of PLD2 mRNA was detected by qRT-PCR. (b) Colonic biopsies were collected from patients with A-CD ( n = 21), R-CD ( n = 27), A-UC ( n = 26), R-UC ( n = 26), and HC ( n = 18). Expression of PLD2 mRNA was examined by qRT-PCR. Gene expression was normalized to GAPDH in each group. ∗∗ p < 0.01 and ∗∗∗ p < 0.001 versus HC. ((c) and (d)) Expression of PLD2 mRNA in inflamed and healthy intestinal mucosa from the same patients with A-CD ((c) n = 14) and A-UC ((d) n = 17) was examined by qRT-PCR. Gene expression was normalized to GAPDH in each group. ∗∗ p < 0.01 and ∗∗∗ p < 0.001 versus unaffected mucosa. (e) Representative images of immunohistochemical staining of PLD2 in inflamed colon from patients with A-CD, A-UC, and normal colonic mucosa from HC. Original magnification ×200 (top) and original magnification ×400 (bottom).

Article Snippet: One group of mice was administered with PLD2 selective inhibitor (CAY10694, Santa Cruz Biotechnology, 4 mg/kg) daily by oral gavage, and another group of mice were administered with PBS as controls.

Techniques: Expressing, Quantitative RT-PCR, Gene Expression, Immunohistochemical staining, Staining

Journal: Mediators of Inflammation

Article Title: Blockade of PLD2 Ameliorates Intestinal Mucosal Inflammation of Inflammatory Bowel Disease

doi: 10.1155/2016/2543070

Figure Lengend Snippet: PLD2 is mainly expressed in neutrophils. (a) Expression of PLD2 in different subsets of immune cells. Peripheral neutrophils, CD20 + B cells, CD8 + T cells, CD4 + T cells, and CD14 + monocytes (1 × 10 6 ) were isolated from healthy donors ( n = 10), and expression of PLD2 was detected by qRT-PCR. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 versus CD14 + monocytes. (b) Neutrophils were isolated from peripheral blood of patients with A-CD ( n = 26), R-CD ( n = 23), A-UC ( n = 26), R-UC ( n = 19), and HC ( n = 20). Expression of PLD2 mRNA was examined by qRT-PCR. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 versus HC. Gene expression was normalized to GAPDH in each group.

Article Snippet: One group of mice was administered with PLD2 selective inhibitor (CAY10694, Santa Cruz Biotechnology, 4 mg/kg) daily by oral gavage, and another group of mice were administered with PBS as controls.

Techniques: Expressing, Isolation, Quantitative RT-PCR, Gene Expression

Journal: Mediators of Inflammation

Article Title: Blockade of PLD2 Ameliorates Intestinal Mucosal Inflammation of Inflammatory Bowel Disease

doi: 10.1155/2016/2543070

Figure Lengend Snippet: TNF- α upregulates PLD2 expression in neutrophils. (a) Neutrophils isolated from healthy donors ( n = 10) were stimulated with IL-6 (10 ng/mL), IL-17A (10 ng/mL), TNF- α (10 ng/mL), IFN- γ (10 ng/mL), LPS (10 ng/mL), and IL-1 β (10 ng/mL), respectively, for 4 h. Expression of PLD2 mRNA was detected by qRT-PCR. ∗ p < 0.05, ∗∗ p < 0.001, and ∗∗∗ p < 0.01 versus medium alone. (b) Neutrophils isolated from healthy donors ( n = 10) were stimulated in vitro with different concentrations of TNF- α as indicated for 4 h; expression of PLD2 was detected by qRT-PCR. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 versus that under culture in medium alone. (c) Neutrophils isolated from healthy donors ( n = 10) were stimulated with TNF- α (10 ng/mL) for 2, 4, and 6 h, and expression of PLD2 was detected by qRT-PCR. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 versus that at the beginning of stimulation. Gene expression was normalized to GAPDH in each group.

Article Snippet: One group of mice was administered with PLD2 selective inhibitor (CAY10694, Santa Cruz Biotechnology, 4 mg/kg) daily by oral gavage, and another group of mice were administered with PBS as controls.

Techniques: Expressing, Isolation, Quantitative RT-PCR, In Vitro, Gene Expression

Journal: Mediators of Inflammation

Article Title: Blockade of PLD2 Ameliorates Intestinal Mucosal Inflammation of Inflammatory Bowel Disease

doi: 10.1155/2016/2543070

Figure Lengend Snippet: Anti-TNF therapy downregulates PLD2 expression. (a) Patients with A-CD ( n = 17) were treated with anti-TNF mAb (IFX, 5 mg/kg) as indicated. Intestinal mucosal biopsies were collected before and at week 12 after the first infusion. Expression of PLD2 mRNA in intestinal mucosa was detected by qRT-PCR. ∗∗ p < 0.01 versus that before IFX treatment. ((b) and (c)) Fresh colonic biopsies were harvested from inflamed mucosa in patients with active CD ((b) n = 10) or active UC ((c) n = 10) and cultured in vitro with IFX or control human IgG (HIg) (50 μ g/mL) for 24 h. Expression of PLD2 mRNA was detected by qRT-PCR. ∗∗ p < 0.01 and ∗∗∗ p < 0.001 versus controls.

Article Snippet: One group of mice was administered with PLD2 selective inhibitor (CAY10694, Santa Cruz Biotechnology, 4 mg/kg) daily by oral gavage, and another group of mice were administered with PBS as controls.

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

Journal: Mediators of Inflammation

Article Title: Blockade of PLD2 Ameliorates Intestinal Mucosal Inflammation of Inflammatory Bowel Disease

doi: 10.1155/2016/2543070

Figure Lengend Snippet: Blockade of PLD2 alleviates DSS-induced colitis in mice. DSS-induced colitis in C57BL/6 mice was induced as indicated. Two groups of DSS-exposed mice ( n = 10) were treated with PLD2 inhibitor (CAY10594, 4 mg/Kg) or PBS as controls daily by oral gavage. Two groups of none DSS-exposed mice ( n = 10) were also treated with CAY10594 or PBS as negative controls. (a) The survival rates of mice after DSS exposure over 10 days. (b) The changes of body weight were observed and expressed as a percentage of initial body weight at the start of experiments during 10-day observation. (c) Gross morphology of colonic tissues on day 10 after DSS induction. (d) The statistical length of colons in different groups. ∗ p < 0.05 and ∗∗ p < 0.01 versus controls. (e) Representative H&E staining images of colonic sections (×200). (f) The changes in pathological scores from colonic sections were calculated as indicated. ∗ p < 0.05 and ∗∗ p < 0.01 versus controls.

Article Snippet: One group of mice was administered with PLD2 selective inhibitor (CAY10694, Santa Cruz Biotechnology, 4 mg/kg) daily by oral gavage, and another group of mice were administered with PBS as controls.

Techniques: Staining

Journal: Mediators of Inflammation

Article Title: Blockade of PLD2 Ameliorates Intestinal Mucosal Inflammation of Inflammatory Bowel Disease

doi: 10.1155/2016/2543070

Figure Lengend Snippet: Cytokines profiles in colonic tissues from DSS-induced colitis mice after PLD2 inhibition. ((a)–(f)) Colonic tissues were obtained from mice on day 10 after DSS-induced colitis, and total RNA was extracted to detect mRNA levels of various cytokines by qRT-PCR. ∗ p < 0.05 and ∗∗ p < 0.01 versus controls. ((g)–(j)) Colonic tissues (0.01 g/sample) from mice on day 10 after DSS-induced colitis were cultured ex vivo at 37°C for 24 h; the supernatants were then collected for detection of TNF- α , IL-17A, IL-1 β , and IL-10 by ELISA. ∗ p < 0.05 and ∗∗ p < 0.01 versus controls.

Article Snippet: One group of mice was administered with PLD2 selective inhibitor (CAY10694, Santa Cruz Biotechnology, 4 mg/kg) daily by oral gavage, and another group of mice were administered with PBS as controls.

Techniques: Inhibition, Quantitative RT-PCR, Cell Culture, Ex Vivo, Enzyme-linked Immunosorbent Assay

Journal: Mediators of Inflammation

Article Title: Blockade of PLD2 Ameliorates Intestinal Mucosal Inflammation of Inflammatory Bowel Disease

doi: 10.1155/2016/2543070

Figure Lengend Snippet: Inhibition of PLD2 promotes neutrophil migration. (a) Bone marrow cells were isolated from DSS-induced mice on day 10, and expression of Ly6G and CD11b was analyzed by flow cytometry. (b) Percentages of Ly6G + CD11b + neutrophils were calculated. ∗ p < 0.05 and ∗∗ p < 0.01 versus controls. ((c) and (d)) Neutrophils were isolated from the bone marrow of mice; expression of CXCR2 (c) and GRK2 (d) was examined by qRT-PCR. ∗ p < 0.05 and ∗∗ p < 0.01 versus controls. (e) Neutrophils were isolated from the bone marrow of mice on day 10 after DSS-induced colitis and stimulated in vitro with PLD2 inhibitor (CAY10594, 10 μ m) for 30 min; neutrophil migration was then analyzed by a Transwell plate (5 μ m) under the stimulation with CXCL1 (0, 50, 100, 200 ng/mL) for 30 min. ∗ p < 0.05 and ∗∗ p < 0.01 versus controls.

Article Snippet: One group of mice was administered with PLD2 selective inhibitor (CAY10694, Santa Cruz Biotechnology, 4 mg/kg) daily by oral gavage, and another group of mice were administered with PBS as controls.

Techniques: Inhibition, Migration, Isolation, Expressing, Flow Cytometry, Quantitative RT-PCR, In Vitro