Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Microtubule Engagement with Taxane Is Altered in Taxane-Resistant Gastric Cancer

doi: 10.1158/1078-0432.CCR-19-3018

Figure Lengend Snippet: Differential gene expression analysis of sensitive and resistant cells reveals coordinated modulation of kinesin family is differentially modulated in docetaxel (DTX)-sensitive and docetaxel-resistant DIF-GC cells, which display characteristic microtubule dynamicity. A, Venn diagram representing the DEGs after docetaxel treatment in docetaxel-sensitive (AZ521 and TMK1) and docetaxel-resistant (SCH and MKN7) gastric cancer cell lines. B, Bar graph representing the microtubule-related pathways identified by Reactome pathway analysis of the 84 common DEGs, ranked by significance. C, mRNA expression of the five kinesins was assessed in biopsies obtained from 12 patients with gastric cancer at baseline, with nine matching on-treatment (cabazitaxel) samples. Fold change of on-treatment versus baseline mRNA expression levels was calculated and correlated to response to treatment. D, Computational analysis of microtubule dynamic parameters by direct detection and tracking of GFP-EB1 comets using the MATLAB-based plusTipTracker algorithm. Graphic display of the microtubule growth speed (μm × per minute) in TMK1 and HS746T cell lines at baseline and after 1 hour treatment with increasing concentrations of docetaxel. Mann–Whitney test was used for statistical analysis (*, P < 0.05; **, P < 0.01; n.s., not significant; Ctr, control).

Article Snippet: Image analysis was performed using the MATLAB-based plusTipTracker algorithm, as described previously ( 21 ).

Techniques: Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: Gαi2-Mediated Regulation of Microtubules Dynamics and Rac1 Activity Orchestrates Cranial Neural Crest Cell Migration in Xenopus

doi: 10.1101/2023.09.07.556733

Figure Lengend Snippet: A-B. Immunofluorescence against α-tubulin. Pseudocolor scale shows that Gαi2MO increases microtubules distribution towards the leading edge. Magnification: 63X. C-D. Tubulin 3D reconstruction in control and Gαi2 knockdown conditions. E. Quantification of tubulin fluorescence intensity. Gαi2 dramatically increased tubulin signal towards the leading edge. Error bars: s.e.m. *** P≤0.001. n= 3 cells were analyzed per condition for each of three independent experiments. F. Schematic representation of the tubulin distribution measurements from the edge of the nucleus to the cell cortex. T: tubulin. G-H. Images were obtained every 1.5 seconds for a total of 5 minutes using Leica SP8 confocal microscopy. Videos of EB3GFP-labeled cells were cropped to a region of interest, and they were analyzed using plusTipTracker software to detect microtubule growing tips via +TIP tracking proteins . Magnification: 63X. I. Cells were separated into four groups based on growth rate (slow < 11 µm/min < fast) and duration (short < 23 sec < long). Cells injected with Gαi2MO present a higher percentage of slow microtubules, and a lower percentage of fast microtubules compared to control cells. J. The average growth rate of Gαi2 morphant microtubules is 8.52 µm/min ± 0.301 and is significantly lower compared to control cells, whose growth rate is on average 10.53 µm/min ± 0.297. ANOVA statistical analysis for multiple conditions and Student’s T-Test (two-tailed) were performed. N = 3 experiments per condition. K. The quantification of the arrests (pauses) of the comets did not show significant differences between control cells and morphant cells for Gαi2. L. Using TrackMate plugin of ImageJ, the trajectory of comets was tracked in time, specifically those that enter or leave a pre-established region of interest at the cell-cell contact and at the leading edge. At the end of the 2.5 minutes, a relationship was made between the comets that remain in the area versus the total comets that entered the area. Control cells show a 53.8% of catastrophes at the leading edge and 75.1% at cell-cell contact. On the other hand, Gαi2 morphant cells show 42.9% of catastrophes at the leading edge versus only 47.3% at the cell-cell contact.

Article Snippet: We conducted time-lapse tracking of EB3-GFP comets at both 3-second and 1.5-second intervals and quantified various parameters, including growth speed, growth length, comet lifetime, and pauses, using automated analysis with the PlusTipTracker software for Matlab ( ; ).

Techniques: Immunofluorescence, Control, Knockdown, Fluorescence, Confocal Microscopy, Labeling, Software, Injection, Two Tailed Test