Journal: BMC Cancer

Article Title: Plocabulin, a novel tubulin-binding agent, inhibits angiogenesis by modulation of microtubule dynamics in endothelial cells

doi: 10.1186/s12885-018-4086-2

Figure Lengend Snippet: Effects of plocabulin on HUVEC cell morphology and microtubule mass by fluorescence microscopy. a HUVEC endothelial cells were cultured in the absence or presence of plocabulin 0.1 nM at different time intervals. Cells were then stained for α-tubulin (red) and nuclei (blue). b HUVEC endothelial cells were cultured in the absence or presence of increasing concentrations of plocabulin for 24 h. Representative images of each treatment conditions are shown

Article Snippet: For microtubule dynamics, EB3-GFP plasmid was nucleofected using Amaxa technologies with the HUVEC nucleofector kit (Lonza) according to the manufacturers’ protocols.

Techniques: Fluorescence, Microscopy, Cell Culture, Staining

Journal: BMC Cancer

Article Title: Plocabulin, a novel tubulin-binding agent, inhibits angiogenesis by modulation of microtubule dynamics in endothelial cells

doi: 10.1186/s12885-018-4086-2

Figure Lengend Snippet: Effects of plocabulin on microtubule dynamics in HUVEC cells. a EB3-GFP maximum intensity projection for two minutes acquisition with two frames/s in each condition; EB3-GFP was transfected into HUVEC cells as describe in material and methods. b Representative kymographs of EB3 dynamics for each condition. Horizontal and vertical bars represent 1 um and 10 s respectively. c Histograms representing the mean velocity (μm/min) and distance of growth events (μm), as well as the mean catastrophe frequency (min − 1 ) in each condition. Data are shown as mean ± SD. Comparisons between different samples were analyzed by Student’s t test. Differences were considered significant at *** P < 0.001

Article Snippet: For microtubule dynamics, EB3-GFP plasmid was nucleofected using Amaxa technologies with the HUVEC nucleofector kit (Lonza) according to the manufacturers’ protocols.

Techniques: Transfection

Journal: Nature Communications

Article Title: Annexin A1 is a polarity cue that directs mitotic spindle orientation during mammalian epithelial morphogenesis

doi: 10.1038/s41467-023-35881-x

Figure Lengend Snippet: a Confocal images of representative metaphase MCF-10A cells transfected with si-Control, si-ANXA1#1 or si-ANXA1#2 stained for α-tubulin (grey) and counterstained with DAPI (DNA, magenta). White arrowheads indicate elongated and buckled astral microtubules. Dashed lines outline the cell contour. b Confocal images of representative metaphase MCF-10A cells stably expressing EB3-GFP and transfected with si-Control, si-ANXA1#1 or si-ANXA1#2. Dashed lines outline the cell contour. c Ratio of astral microtubule and EB3-GFP comets length ( I ) in siRNA-transfected cells: astral microtubules (si-Control: n = 31; si-ANXA1#1: n = 43; si-ANXA1#2: n = 45); EB3-GFP comets (si-Control: n = 30; si-ANXA1#1: n = 34; si-ANXA1#2: n = 34). I 1 and l 2 of astral microtubules were measured as depicted on the illustration. One-way ANOVA with Tukey’s test, astral microtubules: ** P = 0.007 and * P = 0.035; EB3-GFP: *** P = 0.001 and *** P = 0.0009. d Relative fluorescence intensities of astral microtubules ( I astral, rel ) in siRNA-transfected cells (si-Control: n = 31; si-ANXA1#1: n = 47; si-ANXA1#2: n = 47). Fluorescence intensities on the spindle ( I spindle ) and total cell ( I total ) were measured as depicted on the illustration. One-way ANOVA with Tukey’s test, P = 0.467 and P = 0.676. e Ratio of pole-to-cortex distance ( d ) in siRNA-transfected cells (si-Control: n = 40; si-ANXA1#1: n = 48; si-ANXA1#2: n = 52). Pole-to-cortex distance d 1 and d 2 were measured as depicted on the illustration. One-way ANOVA with Tukey’s test, ** P = 0.004 and * P = 0.032. f Confocal images of representative metaphase MCF-10A cells transfected with si-Control, si-ANXA1#1 or si-ANXA1#2 stained for F-actin (grey) and counterstained with DAPI (DNA, magenta). g Cortical to cytoplasmic ratio of actin fluorescence intensities ( I cortex / I cytoplasm ) in siRNA-transfected cells (si-Control: n = 30; si-ANXA1#1: n = 30; si-ANXA1#2 n = 30). Fluorescence intensities in the cytoplasm ( I cytoplasm ) and total cell ( I total ) were measured as depicted on the illustration. One-way ANOVA with Tukey’s test, *** P = 0.001 and *** P = 0.0003. h Confocal images of representative MCF-10A cells treated with DMSO (Control) or 1 µM Latrunculin A for 30 min, stained for F-actin (grey) and counterstained with DAPI (DNA, magenta). i Confocal images of representative MCF-10A cells treated with DMSO (Control) or 1 µM Latrunculin A for 30 min, stained for ANXA1, LGN, NuMA or p150 Glued (grey), and counterstained with DAPI (DNA, magenta). j Average cortical and cytoplasmic fluorescence intensity profiles of ANXA1, LGN, NuMA and p150 Glued from metaphase cells (Control: n = 30; Latrunculin A: n = 30). Asterisks indicate the spindle poles. All data are presented as mean ± s.e.m. from 3 independent experiments. n.s. (not significant). arb. units (arbitrary units). All scale bars, 5 µm. Source data are provided as a Source Data file.

Article Snippet: To clone pTK-EB3-GFP, the EB3 sequence was synthesised (Eurofins) and inserted into pTK-GFP digested by AgeI and SacII restriction enzymes (New England Biolabs).

Techniques: Transfection, Staining, Stable Transfection, Expressing, Fluorescence