Journal: Journal of Biological Chemistry

Article Title: ARF6 Activated by the LHCG Receptor through the Cytohesin Family of Guanine Nucleotide Exchange Factors Mediates the Receptor Internalization and Signaling

doi: 10.1074/jbc.m112.362087

Figure Lengend Snippet: FIGURE 4. Effect of SecinH3 (ARF GEF inhibitor), Wortmannin (PI3K inhibitor), and Gallein (G inhibitor) on HCG-induced HLHCGR internalization and cAMP accumulation. A, HEK-HLHCGR cells pre-incubated with either SecinH3 (1–60 M, E, ●), Wortmannin (0.001–1 M, f, ) or Gallein (0.4–50 M, , Œ) for 15 min (wortmannin and Gallein) or 2 h (SecinH3) at 37 °C were incubated without (open symbols) or with (solid symbols) 10 IU/ml HCG for 30 min at 37 °C and receptor internalization determined by ELISA. B, immunofluorescence analysis of the effect of SecinH3, Wortmannin and Gallein on HCG-stimulated (10 IU/ml HCG, 30 min) HLHCGR internalization. C, cAMP accumulation in unstimulated (open symbols) or HCG-stimulated (solid symbols; 10 IU/ml HCG, 30 min) HEK-HLHCGR cells pretreated with the indicated concentrations of SecinH3, Wortmannin, or Gallein as described in part A above.

Article Snippet: Phamacological inhibitors SecinH3 andQS11were obtained fromMerck whereas Wortmannin, Gallein, and Dynasore were from Tocris.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Immunofluorescence

Journal: Journal of Biological Chemistry

Article Title: ARF6 Activated by the LHCG Receptor through the Cytohesin Family of Guanine Nucleotide Exchange Factors Mediates the Receptor Internalization and Signaling

doi: 10.1074/jbc.m112.362087

Figure Lengend Snippet: FIGURE 5. Effect of SecinH3, Wortmannin and Gallein on ARF6 activation in HCG-stimulated HEK-HLHCGR cells. HCG-stimulated (10 IU/ml HCG, 30 min) ARF6 activation was assessed by a GST-GGA3 PBD pulldown assay in HEK-HLHCGR cells preincubated with the indicated concentrations of (A) SecinH3, (B) Wortmannin, or (C) Gallein. Total ARF6 levels in the cell lysate and the resin bound ARF6-GTP were visualized by immunoblot analysis with an anti-ARF6 mouse mAb. Densitometric analysis of ARF6-GTP is shown as a histogram after normalizing to the expression of total ARF6 present in the sample. For Wortmannin and Gallein, the effect of the drug vehicle (0.5% DMSO) on ARF6-GTP levels, was also determined. Solid bars indicate measurements made in the presence of HCG.

Article Snippet: Phamacological inhibitors SecinH3 andQS11were obtained fromMerck whereas Wortmannin, Gallein, and Dynasore were from Tocris.

Techniques: Activation Assay, Western Blot, Expressing

Journal: Journal of Biological Chemistry

Article Title: ARF6 Activated by the LHCG Receptor through the Cytohesin Family of Guanine Nucleotide Exchange Factors Mediates the Receptor Internalization and Signaling

doi: 10.1074/jbc.m112.362087

Figure Lengend Snippet: FIGURE 6. Effect of various inhibitors on cytohesin 2 translocation and HLHCGR internalization in HCG-stimulated HEK-HLHCGR cells. Immuno- fluorescence analysis of GFP-cytohesin 2 translocation and receptor internal- ization in HCG-stimulated (10 IU/ml HCG, 30 min) HEK-HLHCGR cells pre- treated for 15 min (Wortmannin, Gallein, and Dynasore) or 2 h (SecinH3) at 37 °C without or with the indicated inhibitor or co-transfected with EP15(DN) or NM23-H1 (DN) plasmid. Red indicates anti-Myc antibody staining of Myc- HLHCGR, green indicates GFP, and yellow indicates overlay of red and green. Note that HCG-induced receptor internalization was inhibited by all drug treatments and co-transfection with the indicated DN plasmid, whereas HCG- induced GFP-cytohesin 2 translocation was inhibited by Gallein and Wort- mannin, but not by SecinH3, Dynasore, EPS15 DN, NM23-H1 DN, and PBP10.

Article Snippet: Phamacological inhibitors SecinH3 andQS11were obtained fromMerck whereas Wortmannin, Gallein, and Dynasore were from Tocris.

Techniques: Translocation Assay, Fluorescence, Transfection, Plasmid Preparation, Staining, Cotransfection

Journal: The Journal of Cell Biology

Article Title: Podosome assembly is controlled by the GTPase ARF1 and its nucleotide exchange factor ARNO

doi: 10.1083/jcb.201605104

Figure Lengend Snippet: ARF1-GTP levels and podosome formation. (A) Quantification of ARF1-GTP levels by G-LISA assay in control, stimulated, and inhibitor-treated THP1 cells. Both TGFβ1 and PMA increased the fraction of GTP-bound ARF1 compared with control, whereas treatment with SecinH3 or BFA dramatically reduced it. Pooled results of three independent experiments are shown. Mean ± SD is indicated. (B) Disruption of podosomes labeled with mCherry-Utrophin (UtrCH) upon treatment with SecinH3 (top). Note that integrity of the Golgi apparatus labeled with GFP-mannosidase II was preserved in the same SecinH3-treated cell (bottom). See also Video 3. (C and D) Disruption of podosomes labeled with mCherry-vinculin by SecinH3 (C, top) and BFA (D, top). Although the effect of SecinH3 in these cells was not accompanied by changes in localization of ARF1 to the Golgi and cytoplasmic puncta (C, bottom), BFA disrupted both Golgi and ARF1 puncta (D, bottom); Bars, 5 µm. Insets (1 × 1 µm 2 ) show evolution of individual ARF1 puncta in each case. (E and F) Quantification of the effect of SecinH3 and BFA on number of podosomes per cell (E) and percentage of cells with more than 10 podosomes (F). (G) ARF1-GTP level increase in fibroblasts plated on a RGD-functionalized fluid lipid bilayer compared with fibroblasts plated on glass coverslip. Mean ± SD is indicated. (H) Effect of SecinH3 on the integrity of podosome-like structures formed by fibroblasts plated on fluid lipid bilayer. (I) Quantification of the disruptive effect of SecinH3 and BFA on podosome-like structures formed by fibroblasts on lipid bilayer. The percentage of podosome-forming cells significantly decreased upon treatment by each of the inhibitors. The data in E, F, and I are presented as indicated in the legend to . Pooled data of three independent experiments are presented for each group. The significance of the difference between groups was estimated by two-tailed Student’s t test. ns, P > 0.05 (nonsignificant); *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

Article Snippet: For drug inhibition studies, cells were treated with 30 μM SecinH3 (Tocris Bioscience), 10 μM Golgicide A (Santa Cruz Biotechnology, Inc.), 5 μg/ml BFA (Sigma-Aldrich), 30 μM Y-27632 (Sigma-Aldrich), 25 μM GM6001 (Enzo Life Sciences), and 2 μg/ml C3 transferase (Cytoskeleton, Inc.) in complete medium for 1–2 h or 4 h for GM6001 at 37°C with 5% CO 2 and subsequently fixed with 4% PFA.

Techniques: Control, Disruption, Labeling, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: Podosome assembly is controlled by the GTPase ARF1 and its nucleotide exchange factor ARNO

doi: 10.1083/jcb.201605104

Figure Lengend Snippet: Inhibition of ARF1 activity induces RhoA activation. (A–C) 1-h incubation of TGFβ1-stimulated THP1 cells with 30 µM SecinH3 led to increase in RhoA-GTP (A) but not Rac1-GTP (B) or Cdc42-GTP (C) fractions as indicated by Western blots after pull-down assay. (D–J) SIM visualization of podosome dynamics in TGFβ1-stimulated THP1. (D and E) Live imaging of cell stably transfected with GFP-MRLC to visualize myosin-II filaments and RFP-Lifeact to visualize podosome cores. (D) Cell treated with 30 µM SecinH3 shows an increase in myosin-IIA filament assembly (green) and disruption of podosomes (red). Enlarged image of white-boxed area (5 × 4.5 µm 2 ) of D shows colocalization between appearance of myosin-IIA filaments and podosome disruption (see Video 7). (E) Time course of podosome reappearance after addition of 30 µM ROCK inhibitor Y-27632 to cell incubated in SecinH3-containing medium. Note that podosomes (red) appeared after disassembly of myosin-II filaments (green). (F–J) TGFβ1-stimulated THP1 cells were transfected with either control scrambled siRNA (F and G) or with siRNA to NM-myosin-IIA heavy chain, MYH9 (I and J) and, after 48 h, treated with either 0.1% DMSO (F and I) or 30 µM SecinH3 (G and J) for 1 h. (F, G, I, and J) After fixation, the cells were stained with phalloidin (left) and antibodies to NM-myosin-IIA heavy chain (middle) and vinculin (right). Bars, 5 µm. (H) Western blot showing protein levels of NM-myosin-IIA heavy chain in control cells (transfected with scrambled siRNA) or in NM-myosin-IIA knockdown cells (transfected with MYH9 siRNA); α-tubulin was used as a loading control. (K and L) Effect of SecinH3 treatment of control and NM-myosin-IIA knockdown cells on number of podosomes per cell (K) and percentage of cells with more than 10 podosomes (L). The data in K and L are presented as indicated in the legend to . Pooled data of at least two independent experiments are presented for each group. The significance of the difference between groups was estimated by two-tailed Student’s t test. nonsignificant (ns), P > 0.05; ***, P ≤ 0.001.

Article Snippet: For drug inhibition studies, cells were treated with 30 μM SecinH3 (Tocris Bioscience), 10 μM Golgicide A (Santa Cruz Biotechnology, Inc.), 5 μg/ml BFA (Sigma-Aldrich), 30 μM Y-27632 (Sigma-Aldrich), 25 μM GM6001 (Enzo Life Sciences), and 2 μg/ml C3 transferase (Cytoskeleton, Inc.) in complete medium for 1–2 h or 4 h for GM6001 at 37°C with 5% CO 2 and subsequently fixed with 4% PFA.

Techniques: Inhibition, Activity Assay, Activation Assay, Incubation, Western Blot, Pull Down Assay, Imaging, Stable Transfection, Transfection, Disruption, Control, Staining, Knockdown, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Gβγ translocation to the Golgi apparatus activates ARF1 to spatiotemporally regulate G protein–coupled receptor signaling to MAPK

doi: 10.1016/j.jbc.2021.100805

Figure Lengend Snippet: Effects of ARF1 inhibitors on the activation and GA recruitment of ARF1 induced by SDF1α. A , effects of ARF1 inhibitors on ARF1 activation. The cells were treated with secinH3 (100 μM), GCA (30 μM), or Exo2 (60 μM) for 30 min before stimulation with SDF1α at 200 ng/ml for 5 min. B , dose-dependent effect of Exo2 on ARF1 activation by SDF1α. C , quantitative data shown in B (n = 3). D , effects of ARF1 inhibitors on ARF1 localization at the GA. The cells were treated with individual inhibitors before SDF1α stimulation as mentioned previously, and ARF1 localization at the GA was visualized by confocal imaging following staining with antibodies against ARF1 and p230. E , quantitative data showing ARF1 expression at the GA relative to its total expression (n = 50 cells in three experiments). The quantitative data shown in C and E are presented as means ± SD. The images shown in each panel are representatives of at least three experiments. ∗ p < 0.05 versus respective control. The scale bar represents 10 μm. ARF1, ADP-ribosylation factor 1; GA, Golgi apparatus; GCA, golgicide A; SDF1α, stromal cell–derived factor 1α.

Article Snippet: Human SDF1α was purchased from PeproTech; UK14304, rapamycin, brefeldin A, monensin, nigericin, swainsonine, and Exo2 were from Sigma–Aldrich; pertussis toxin was from List Biological Laboratories; gallein was from Tocris Bioscience; wortmannin, AS-604850, and GSK2292767 were from ApexBio; TGX-221 and HS-173 were from Adooq Bioscience; secinH3, GCA, nocodazole, ilimaquinone, AMD3100, control siRNA, and siRNA targeting human ARF1, CRISPR–Cas9 control plasmids, and knockout plasmids targeting human ARF1, and antibodies against phospho-ERK1/2 and β-actin were from Santa Cruz Biotechnology; p230 antibodies were from BD Transduction Laboratories; ARF1 antibodies were purchased from Abcam; ERK1/2 antibodies were from Cell Signaling Technology; 12-well inserts and Matrigel matrix were from Corning.

Techniques: Activation Assay, Imaging, Staining, Expressing, Control, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Gβγ translocation to the Golgi apparatus activates ARF1 to spatiotemporally regulate G protein–coupled receptor signaling to MAPK

doi: 10.1016/j.jbc.2021.100805

Figure Lengend Snippet: Effects of ARF1 inhibitors on ERK1/2 activation by SDF1α and Golgi-Gγ9. A , effect of ARF1 inhibitors on ERK1/2 activation by SDF1α. The cells were treated with secinH3 (100 μM), GCA (30 μM), or Exo2 (60 μM) for 30 min before stimulation with SDF1α at 200 ng/ml for 5 min. B , effect of ARF1 inhibitors on ERK1/2 activation by Golgi-Gγ9. The cells were transfected with FRB-γ9, Gβ1, and Golgi-FKBP (500 ng each) and then incubated with individual inhibitors before incubation with rapamycin for 30 min. The Western blots shown in each panel are representatives of at least three experiments. ARF1, ADP-ribosylation factor 1; ERK1/2, extracellular signal–regulated protein kinases 1 and 2; FKBP, FK506-binding protein; FRB, FKBP–rapamycin binding; GCA, golgicide A; SDF1α, stromal cell–derived factor 1α.

Article Snippet: Human SDF1α was purchased from PeproTech; UK14304, rapamycin, brefeldin A, monensin, nigericin, swainsonine, and Exo2 were from Sigma–Aldrich; pertussis toxin was from List Biological Laboratories; gallein was from Tocris Bioscience; wortmannin, AS-604850, and GSK2292767 were from ApexBio; TGX-221 and HS-173 were from Adooq Bioscience; secinH3, GCA, nocodazole, ilimaquinone, AMD3100, control siRNA, and siRNA targeting human ARF1, CRISPR–Cas9 control plasmids, and knockout plasmids targeting human ARF1, and antibodies against phospho-ERK1/2 and β-actin were from Santa Cruz Biotechnology; p230 antibodies were from BD Transduction Laboratories; ARF1 antibodies were purchased from Abcam; ERK1/2 antibodies were from Cell Signaling Technology; 12-well inserts and Matrigel matrix were from Corning.

Techniques: Activation Assay, Transfection, Incubation, Western Blot, Binding Assay, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Gβγ translocation to the Golgi apparatus activates ARF1 to spatiotemporally regulate G protein–coupled receptor signaling to MAPK

doi: 10.1016/j.jbc.2021.100805

Figure Lengend Snippet: Inhibition and depletion of ARF1 suppress PC3 migration and invasion induced by SDF1α. A , inhibition of PC3 migration and invasion by ARF1 and PI3Kγ inhibitors as measured in transwell assays. PC3 cells were treated with SDF1α at 1 μg/ml together with secinH3 (100 μM), GCA (30 μM), Exo2 (60 μM), or AA-604850 (2.5 μM) for 48 h. Stimulation with FBS at 10% was used as a positive control. B , inhibition of PC3 migration and invasion by siRNA-mediated ARF1 knockdown. PC3 cells were transfected with control or ARF1 siRNA and then treated with SDF1α for 48 h. C , inhibition of PC3 migration and invasion by CRISPR–Cas9-mediated depletion of ARF1. PC3 cells were transfected with control or ARF1 knockout plasmids and then treated with SDF1α for 48 h. The quantitative data are presented as means ± SD (n = 10). ∗ and ∗∗ p < 0.005 versus basal and control, respectively. ARF1, ADP-ribosylation factor 1; FBS, fetal bovine serum; GCA, golgicide A; SDF1α, stromal cell–derived factor 1α.

Article Snippet: Human SDF1α was purchased from PeproTech; UK14304, rapamycin, brefeldin A, monensin, nigericin, swainsonine, and Exo2 were from Sigma–Aldrich; pertussis toxin was from List Biological Laboratories; gallein was from Tocris Bioscience; wortmannin, AS-604850, and GSK2292767 were from ApexBio; TGX-221 and HS-173 were from Adooq Bioscience; secinH3, GCA, nocodazole, ilimaquinone, AMD3100, control siRNA, and siRNA targeting human ARF1, CRISPR–Cas9 control plasmids, and knockout plasmids targeting human ARF1, and antibodies against phospho-ERK1/2 and β-actin were from Santa Cruz Biotechnology; p230 antibodies were from BD Transduction Laboratories; ARF1 antibodies were purchased from Abcam; ERK1/2 antibodies were from Cell Signaling Technology; 12-well inserts and Matrigel matrix were from Corning.

Techniques: Inhibition, Migration, Positive Control, Knockdown, Transfection, Control, CRISPR, Knock-Out, Derivative Assay

Journal: Scientific Reports

Article Title: In PC3 prostate cancer cells ephrin receptors crosstalk to β 1 -integrins to strengthen adhesion to collagen type I

doi: 10.1038/srep08206

Figure Lengend Snippet: Graph depicts the adhesion force of PC3 cells after being in contact with collagen I for 60 s in the presence of inhibitors; wortmannin (100 nM, 0.01% final DMSO concentration), LY294002 (50 μM, 0.5%), NSC23766 (50 μM, 0.1%) and EHT1864 (100 μM, 0.13%), SecinH3 (20 μM, 0.1%), Akt inhibitor IV (1 μM, 0.1%), Akt inhibitor VIII (20 μM, 0.1%), GGTI2147 (10 μM, 1%), GGTI286 (10 μM, 1%), blebbistatin (10 μM, 0.02%), CTO4 (2 μg/mL, 1% glycerol), and Y27632 (10 μM, 0.1%). Blue and brown data points represent cells bound to ConA and ephrin-A1 coated cantilevers, respectively. Adhesion values recorded at contact times of 5 and 15 s are given in . Each dot represents the measurement of one PC3 cell. The number of cells assayed for each condition is given by . Bars mark mean force and standard deviation. Statistical differences to control experiments (untreated cells) were analyzed by Mann-Whitney U -tests ( P -values are given). Perturbations were considered significant if all inhibitors targeting the same protein significantly reduced the adhesion force.

Article Snippet: To inhibit cell signaling, wortmannin (100 nM; Sigma-Aldrich) , LY294002 (50 μM; Cell Signaling Technology) , NSC23766 (50 μM; Merck Millipore) , EHT 1864 (100 μM; R&D Systems) , GGTI 2147 or GGTI286 (10 μM; Merck Millipore) , Akt inhibitor IV (1 μM; Merck Millipore) , Akt inhibitor VIII (20 μM; Merck Millipore) , SecinH3 (20 μM; Merck Millipore) , blebbistatin (10 μM; Merck Millipore) or Y-27632 (10 μM; Merck Millipore) were added to the measurement media at the concentrations given.

Techniques: Concentration Assay, Standard Deviation, MANN-WHITNEY

Journal: Nature cell biology

Article Title: An ARF6-Exportin-5 Axis Delivers pre-miRNA Cargo to Tumor Microvesicles.

doi: 10.1038/s41556-019-0345-y

Figure Lengend Snippet: a. Exportin-5 localization was examined by immunofluorescence in the presence of SecinH3. Scale bar = 15 μm. Representative images (N=5 biologically independent experiments shown. b. Exportin-5 western blot from equal numbers of TMVs from control or SecinH3 treated cells. Data presented as mean±SD for N=4 biologically independent experiments. P-value calculated using unpaired two-tailed t-test. c. Melanoma cells were co-treated with SecinH3 and chloroquine, and the intracellular distribution of Exportin-5 examined by confocal microscopy. Scale bar = 15 μm. Images representative of N=3 biologically independent experiments. d. Predicted interaction between Exportin-5 and ARF6 with GRP1 (4kax) was modeled, highlighting the energetically favorable binding arrangements listed in e . f. Exportin-5 co-immunoprecipitation from melanoma cells was analyzed by western blot to confirm the inclusion of GRP1 in complex with Exportin-5 and ARF6. Blots represent N=3 biologically independent experiments. g. Endogenous GRP1 was depleted from melanoma cells prior to immunoprecipitation of HA-ARF6. Co-precipitating proteins were separated by SDS-PAGE and analyzed by western blotting as indicated. Representative blots (N=3 biologically independent experiments) shown. h. Levels of Exportin-5, Dicer, and Argonaute-2 TMV cargo were analyzed by western blotting following isolation from cells depleted of endogenous GRP1. Data presented as mean±SD (N=5 biologically independent samples). P-value determined by unpaired two-tailed t-test. i, j. TMV pre-miRNA content was isolated from cells expressing 2 independent shRNA hairpins against GRP1. Isolated RNA was then analyzed by qRT-PCR. For each condition Data presented as mean±SD from 3 biologically independent experiments. P-values determined by unpaired, two-tailed t-test between control and treatment reactions for each independent pre-miRNA amplification reaction. k. Equal numbers of TMVs (1×10 8 ) were isolated from control or GRP1-shRNA treated LOX ARF6-Q67L cells and lysates analyzed by western blotting as indicated. l. ARF6 was immunoprecipitated from LOX ARF6-Q67L cells with or without GRP1-shRNA. Co-precipitating proteins were examined by western blot as indicated. For panels k-l representative blots (N=3 biologically independent experiments) shown. In all panels, p-values <0.05 were considered significant. Unprocessed blot images shown in . Statistical Source in .

Article Snippet: SecinH3 and TBB were purchased from Cayman Chemical.

Techniques: Immunofluorescence, Western Blot, Control, Two Tailed Test, Confocal Microscopy, Binding Assay, Immunoprecipitation, SDS Page, Isolation, Expressing, shRNA, Quantitative RT-PCR, Amplification