Journal: Traffic (Copenhagen, Denmark)

Article Title: The Rab27a Effectors JFC1/Slp1 and Munc13-4 Regulate Exocytosis of Neutrophil Granules

doi: 10.1111/j.1600-0854.2008.00838.x

Figure Lengend Snippet: Dual-color live cell imaging to simultaneously visualize the colocalization of JFC1/Slp1 and Rab27a in a spatio-temporal manner in granulocytes. HL-60 granulocytes were cotransfected with vectors for the expression of EGFP-Rab27 and DsRED-JFC1 and analyzed by TIRFM. Laser illumination was adjusted to impinge on the coverslip at an angle to yield a calculated evanescent field depth of 108 nm. Images were analyzed using Imaris software. A) Rab27a (green), JFC1 (red) and overlaid (merged) images showing true colocalization of Rab27a and JFC1 on most vesicles. The dynamics of the labeled vesicles and the spatio-temporal displacement of JFC1 and Rab27a were followed for 1 min and can be viewed in associated Movies S1–S3. B) Analysis of vesicle dynamics using imaris software. Left panel, each sphere represents a vesicle. Right panel, the tracks indicate the trajectory of the associated vesicles occurring during the analysis. Increasing colour in a given track is indicative of time of appearance and residence in the TIRF zone. Tracks without a corresponding vesicle indicate that the vesicle appeared in the TIRF zone at a different frame from that shown in (A). Vesicles without associated tracks represent docked vesicles. The dynamics of these vesicles during the length of the analysis can be observed in associated Movie S4 and can be found in the data analysis chart (Table S1). All vesicles (n = 254) that appeared in the TIRFM zone during the length of the study (1 min) were included in the analysis. C) HL-60 granulocytes were transfected with a vector for the expression of EGFP-Rab27a and analyzed by TIRFM. The cells were stimulated with PMA (0.1 µg/mL), and vesicle dynamics were continuously monitored for 7 min. Images were collected at one frame per second. The white arrows indicate a docked vesicle finishing the fusion proces s. The black arrows indicate a vesicle appearing in the TIRFM zone 26 seconds after the addition of the stimulus and subsequently undergoing fusion. Synchronous increase in total intensity and a concomitant increase in the width of fluorescence are consistent with fusion of the vesicle to the plasma membrane (44). Scale bar = 1 µm.

Article Snippet: Movie S4: Analysis of vesicle mobilization in HL-60 cells expressing EGFP-Rab27a and DsRed-JFC1 using imaris software.

Techniques: Live Cell Imaging, Expressing, Software, Labeling, Transfection, Plasmid Preparation, Fluorescence, Membrane

Journal: Traffic (Copenhagen, Denmark)

Article Title: The Rab27a Effectors JFC1/Slp1 and Munc13-4 Regulate Exocytosis of Neutrophil Granules

doi: 10.1111/j.1600-0854.2008.00838.x

Figure Lengend Snippet: Downregulation of JFC1/Slp1 impairs MPO secretion in HL-60 cells. A) Sequences of the gene-targeting shRNA used in this work. The nucleotides of JFC1 targeted by these interference molecules are indicated between parentheses. B) HL-60 cells were stably transfected with three vectors expressing shRNAs specific for JFC1 or with a control empty vector (pRS; OriGene). After differentiation, the expression of JFC1 was analyzed by western blot. PGK1 was used as control for equal loading. C) Expression of Rab27a in JFC1-downregulated cells was verified by western blot. D) Clones 70 and 71 and cells transfected with the control vector (control) were stimulated with 1 µm fMLP (red columns) or left untreated (black columns). Secreted MPO was detected by ELISA. Results represent the mean ± SEM from three independent experiments. The unstimulated control was designated as 100%. *p < 0.03.

Article Snippet: Movie S4: Analysis of vesicle mobilization in HL-60 cells expressing EGFP-Rab27a and DsRed-JFC1 using imaris software.

Techniques: shRNA, Stable Transfection, Transfection, Expressing, Control, Plasmid Preparation, Western Blot, Clone Assay, Enzyme-linked Immunosorbent Assay

Journal: Traffic (Copenhagen, Denmark)

Article Title: The Rab27a Effectors JFC1/Slp1 and Munc13-4 Regulate Exocytosis of Neutrophil Granules

doi: 10.1111/j.1600-0854.2008.00838.x

Figure Lengend Snippet: Interference with JFC1/Slp1 inhibits MPO secretion in permeabilized human neutrophils. A) Electron micrographs of neutrophils permeabilized with SLO (+SLO) or untreated control (−SLO). The upper panel shows that granule distribution is not affected by SLO treatment. Granules appear separated from the plasma membrane by distance of 80–180 nm around the perimeter of both SLO-treated or untreated cells. The lower panel shows that the plasma membrane is not grossly damaged in SLO-treated cells. B) MPO exocytosis was evaluated in SLO-permeabilized 0neutrophils in the presence of 1 mm Mg2+/ATP and 100 µm Ca2+ after incubation with anti-JFC1 or anti-Rab27a antibodies. The cells were stimulated with 1 µm fMLP. Where indicated, the cells were treated with 100 ng/mL LPS before stimulation. Results represent the mean ± SEM from three independent experiments. *p < 0.05 versus control fMLP and **p < 0.05 versus control LPS + fMLP.

Article Snippet: Movie S4: Analysis of vesicle mobilization in HL-60 cells expressing EGFP-Rab27a and DsRed-JFC1 using imaris software.

Techniques: Control, Membrane, Incubation

Journal: Traffic (Copenhagen, Denmark)

Article Title: The Rab27a Effectors JFC1/Slp1 and Munc13-4 Regulate Exocytosis of Neutrophil Granules

doi: 10.1111/j.1600-0854.2008.00838.x

Figure Lengend Snippet: Interference with Rab27a but not JFC1/Slp1 impairs MMP-9 secretion in neutrophils. A) Immunofluorescence analysis of the distribution of Rab27a and MMP-9 in human neutrophils. Endogenous Rab27a was detected using a specific antibody raised in rabbit, and MMP-9 was detected using an antibody raised in goat. The arrows show areas of colocalization in the upper panel. The lower panel shows a magnification of the area of colocalization indicated in the upper panel. B) Immunofluorescence analysis of the distribution of JFC1 and MMP-9 in neutrophils was performed as described in (A). The inset shows lack of colocalization between JFC1 and MMP-9 in most vesicles. The exception is indicated with an arrow. Scale bar = 5 µm. C) MMP-9 exocytosis was evaluated in SLO-permeabilized neutrophils as described under Figure 6. For molecular interference, we used anti-JFC1 or anti-Rab27a antibodies raised in rabbit. Results represent the mean ± SEM from three independent experiments. *p < 0.03 versus control fMLP and **p < 0.05 versus control LPS + fMLP.

Article Snippet: Movie S4: Analysis of vesicle mobilization in HL-60 cells expressing EGFP-Rab27a and DsRed-JFC1 using imaris software.

Techniques: Immunofluorescence, Control

Journal: bioRxiv

Article Title: Hyperosmotic Stress of HL-60/S4 Cells: Gene Set Enrichment Analysis and Microscopic Changes

doi: 10.1101/2025.04.22.649232

Figure Lengend Snippet: In Vivo staining by MitoTracker Red of undifferentiated HL-60/S4 mitochondria in 0 and 300 mM sucrose for 30 min, prior to fixation with HCHO. The yellow asterisks denote the positions of mitotic cells before and after exposure to hyperosmotic stress. Colors: Active Mitochondria (Red); DNA (Cyan). The red staining of mitochondria indicates that they were active in both buffer situations. Magnification bar: 10 μm

Article Snippet: Leica SP8 confocal imaging of HL-60/S4 cells (+/- 300 mM sucrose) employed the following antibodies (including their source and dilution): In Vivo staining of mitochondria activity: MitoTracker Red CMXRos was purchased from Cell Signaling.

Techniques: In Vivo, Staining

Journal: Molecular pharmaceutics

Article Title: An indole-chalcone inhibits multidrug-resistant cancer cell growth by targeting microtubules

doi: 10.1021/acs.molpharmaceut.8b00359

Figure Lengend Snippet: The 72-h cytotoxicity profile of FC77 against the NCI-60 cancer cell line panel.

Article Snippet: Through this screening, FC77 was found to be a low-nanomolar inhibitor of the growth of the majority of the NCI-60 human cancer cell lines (average GI 50 : ~6 nM; and Supplementary Fig. S4 ), with some preference toward leukemia, colon, and central nervous system cancers (GI 50 < 4 nM for all 19 cell lines), as well as toward non-small-cell lung cancers, with only one exception (HOP-92). table ft1 table-wrap mode="anchored" t5 caption a7 Cell line GI 50 (nM) Cell line GI 50 (nM) Cell line GI 50 (nM) Cell line GI 50 (nM) Leukemia Colon cancer CNS cancer A498 2.5 CCRF-CEM 5.1 COLO 205 3.5 SF-268 5.5 ACHN 6.2 Hl-60 (TB) 2.3 HCC-2998 4.3 SF-295 3.0 CAKI-1 4.2 K562 3.5 HCT-116 3.6 SF-539 2.8 RXF 393 N.D. MOLT-4 5.5 HCT-15 3.4 SNB-19 4.1 TK-10 > 100 PRMI-8226 4.8 HT29 4.0 SNB-75 3.3 UO-31 7.9 SR 3.3 KM12 3.6 U251 4.4 Prostate cancer Non-small-cell lung cancer SW-620 4.2 Ovarian cancer PC-3 7.9 A549/ATCC 4.1 Melanoma IGROV1 5.6 DU-145 3.8 EKVX 5.2 LOX IMVI 4.7 OVCAR-3 3.3 Breast cancer HOP-62 5.4 MALME-3M > 100 OVCAR-4 25.1 MCF-7 3.5 HOP-92 74.1 M14 3.4 OVCAR-5 85.1 MDA-MB-231/ATCC 3.9 NCI-H226 6.2 MDA-MB-435 2.3 OVCAR-8 3.5 HS 578T 4.8 NCI-H23 5.1 SK-MEL-2 12.3 NCI/ADR-RES 3.2 BT-549 5.9 NCI-H460 3.7 SK-MEL-28 17.4 SK-OV-3 7.8 T-47D N.D. NCI-H522 2.8 SK-MEL-5 4.3 Renal cancer MDA-MB-468 3.3 UACC-257 > 100 786–0 6.5 Open in a separate window CNS, central nervous system; GI 50 , concentration causing 50% inhibition of the cell growth; NCI, National Cancer Institute; N.D., not determined.

Techniques: