Journal: Science Advances

Article Title: Therapeutic gene silencing of CKAP5 leads to lethality in genetically unstable cancer cells

doi: 10.1126/sciadv.ade4800

Figure Lengend Snippet: ( A ) Representative phenotypes of EB3 localization in control and CKAP5 -silenced groups. NAR cells were transiently transfected with EB3.eGFP plasmid to label the microtubule + ends. After 12 hours of transfection, cells were further transfected with siControl/si CKAP5 LNPs, and live-cell imaging was performed after 30 hours of siRNA-mediated silencing. Live cell imaging was performed by a superresolution spinning disk microscope. Images were captured at 100×. Scale bars, 5 μm. Images were captured every second for a period of 90 s. The images represent superimposition images of 91 frames with each frame marked in a specific shade as shown in the time frame scale. Nondynamic + ends superimpose on each other in all 90 frames due to their static nature and represent as white due to superimposition, whereas dynamic + ends do not superimpose in all the 90 frames due to their dynamic behavior and thus appear as a rainbow color. Thus, higher dynamics of tubulin result in a wider range of colors in these representative superimposed images. ( B ) Quantitative representation of the microtubule (MT) growth speed in control and CKAP5 -silenced group. The live-cell kinetic data obtained from a spinning disk microscope was subjected to Utrack analysis using MATLAB software. ** P = 0.0097. ( C ) Quantitative representation of the MT growth lifetime in control- and CKAP5 -silenced group as measured by MATLAB analysis. ( D ) Quantitative representation of the MT growth length in control and CKAP5 -silenced group as measured by MATLAB analysis. For all the graphical analysis in (B) to (D), the data are represented as mean ± SEM from 10 mitotic events and statistically analyzed by an unpaired t test.

Article Snippet: Application of the Utrack analysis program of the MATLAB to measure the dynamics as well as lifetime of EB3 comets clearly showed a significant reduction in the microtubule growth rate in the CKAP5 -silenced group (1.6-fold reduction, P value 0.0097) ( ).

Techniques: Control, Transfection, Plasmid Preparation, Live Cell Imaging, Microscopy, Software

Journal: Science Advances

Article Title: Therapeutic gene silencing of CKAP5 leads to lethality in genetically unstable cancer cells

doi: 10.1126/sciadv.ade4800

Figure Lengend Snippet: ( A ) Representative phenotypes of EB3 localization in control and CKAP5 -silenced groups. NAR cells were transiently transfected with EB3.eGFP plasmid to label the microtubule + ends. After 12 hours of transfection, cells were further transfected with siControl/si CKAP5 LNPs, and live-cell imaging was performed after 30 hours of siRNA-mediated silencing. Live cell imaging was performed by a superresolution spinning disk microscope. Images were captured at 100×. Scale bars, 5 μm. Images were captured every second for a period of 90 s. The images represent superimposition images of 91 frames with each frame marked in a specific shade as shown in the time frame scale. Nondynamic + ends superimpose on each other in all 90 frames due to their static nature and represent as white due to superimposition, whereas dynamic + ends do not superimpose in all the 90 frames due to their dynamic behavior and thus appear as a rainbow color. Thus, higher dynamics of tubulin result in a wider range of colors in these representative superimposed images. ( B ) Quantitative representation of the microtubule (MT) growth speed in control and CKAP5 -silenced group. The live-cell kinetic data obtained from a spinning disk microscope was subjected to Utrack analysis using MATLAB software. ** P = 0.0097. ( C ) Quantitative representation of the MT growth lifetime in control- and CKAP5 -silenced group as measured by MATLAB analysis. ( D ) Quantitative representation of the MT growth length in control and CKAP5 -silenced group as measured by MATLAB analysis. For all the graphical analysis in (B) to (D), the data are represented as mean ± SEM from 10 mitotic events and statistically analyzed by an unpaired t test.

Article Snippet: EB3 dynamics was quantified by using the utrack MATLAB analysis method as mentioned in the protocol.

Techniques: Control, Transfection, Plasmid Preparation, Live Cell Imaging, Microscopy, Software