Journal: Journal of Extracellular Vesicles

Article Title: Pharmacological inhibition of syntenin PDZ2 domain impairs breast cancer cell activities and exosome loading with syndecan and EpCAM cargo

doi: 10.1002/jev2.12039

Figure Lengend Snippet: SyntOFF regulates the loading of exosomes with syntenin‐dependent cargo and EpCAM. (a) Schematic representation of an exosome highlighting which markers were previously shown to rely on syntenin (red, syntenin pathway) or to be unaffected by syntenin (blue, syntenin independent) for their secretion in MCF7 exosomes. The scheme also represents additional exosome cargo (cargo proteins, among which Fibronectin, EGFR and EpCAM investigated in this study). (b‐d) MCF7 cells were treated with DMSO vehicle (Ctrl) or the indicated compound (CAY10594 10 µM; cSynOFF , C58 , or SyntOFF at 100 µM) for 16 h before analysis. Total cell lysates (See supplementary figure ) and the corresponding exosomes were analyzed by western blot, tracing different markers, as indicated. Histograms represent mean signal intensities (±SEM) in exosomes, relative to controls (Ctrl). Data were obtained from n independent experiments, as indicated. Statistical analysis was performed using the one‐way analysis of variance (ANOVA) with a Bonferroni posttest (* P < 0.05; ** P < 0.01; *** P < 0.001)

Article Snippet: The CAY10594 inhibitor (used at 10 μM) was purchased from Santa Cruz (sc‐223874).

Techniques: Western Blot

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: PLD2 is critical for chemokine-dependent PA generation at the plasma membrane. a (Top) Displacement (left) and velocity (right) of T cells were measured on ICAM-1 with or without CCL21 in the presence or absence of 1 or 2 μM of CAY10593, CAY10594, and 5 or 10 μM of R59022 ( n = 30). * 1 P < 0.002, * 2 P < 0.001 versus WT T cells. (Bottom) Representative tracks of T cells treated with the indicated inhibitors with CCL21 are shown. Each line represents a single-cell track. b (Left upper) Displacement and velocity of scramble (control) or PLD2-knockdown cells were measured on the ICAM-1 with or without CXCL12 ( n = 30). * P < 0.001 versus control cells. (Left lower) Representative tracks of control or PLD2 KD cells with CXCL12 are shown. Each line represents a single-cell track. (Right) BAF cells treated with or without CAY10594 were stimulated with CXCL12 at the indicated times, lysed, and subjected to the pull-down assay. Bound Rap1 (Rap1-GTP) and total Rap1 were detected with anti-Rap1. c PASS-GFP-expressing control cells were stimulated with CXCL12 for the indicated times. Time 0 represents the first time-lapse image; subsequent images were obtained in the same focal plane. Scale bar, 5 μm. d Distribution of PASS-GFP and RA-GEF-1 in BAF cells that were untreated (none) or treated with CXCL12 for 10 min in the presence (middle) or absence (top) of CAY10594 is shown. Scale bar, 5 μm. (Bottom) The graph shows the percentages of cells with the polarized membrane localization of PASS at the plasma membrane ( n = 30). * P < 0.001 versus CXCL12-stimulated cells in the absence of CAY10594. Each bar graph represents the means ± SEM

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 chemokine-dependent Rap1 regulation.

Techniques: Clinical Proteomics, Membrane, Control, Knockdown, Pull Down Assay, Expressing

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: 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 chemokine-dependent Rap1 regulation.

Techniques: Clinical Proteomics, Membrane, Activity Assay, Expressing

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: Oncotarget

Article Title: Subthreshold IKK activation modulates the effector functions of primary mast cells and allows specific targeting of transformed mast cells

doi:

Figure Lengend Snippet: (A, B) BMMCs were pre-treated with BAPTA-AM and stimulated with IL-3 (A) or IL-33 (B) . Lysates were analyzed by westernblotting. (C) BMMCs were pre-treated with BAPTA-AM and single stimulated with IL-33 or IL-33 in combination with IL-3. Supernatants were collected and analyzed for IL-6 ( p < 0,001). (D) BMMCs were pre-treated with different ionomycin concentrations and stimulated with IL-33. Supernatants were collected and analyzed for IL-6. (E–G) BMMCs were pre-treated with BAPTA-AM (E) the IKK-inhibitor VII (F) or SU6656 (G) (E, G; p < 0,001). Cells were single stimulated with IL-33 or IL-33 in combination with ionomycin. Supernatants were collected and analyzed for IL-6. Wt, Pld1 −/− (H) or U73122 (I) -treated BMMCs were single stimulated with IL-33 or IL-33 in combination with IL-3. Supernatants were collected and analyzed for IL-6 ( I ; p < 0,001).

Article Snippet: Furthermore we used the protein biosynthesis inhibitor cyclohexamide (340 μM), the PLD1 inhibitor CAY10594 (10 μM), and the Ca 2+ chelator BAPTA-AM (10 μM) or ionomycin (in a concentration from 0,5–10 ng/ml) (all inhibitors were from Merck Millipore).

Techniques: