CKAP5沉默介导的NAR细胞死亡机制。在所有实验中，细胞分别用siccontrol或siCKAP5-LNPs (0.25 μg/ml)处理。(A)点图显示对照和处理细胞的凋亡和坏死细胞。在指定的时间点收获细胞，PI和膜联蛋白V-APC染色后进行流式细胞术。(B)凋亡细胞死亡的定量测定。数据为三个代表性实验(n = 2)的平均%总体±SEM，采用非配对t检验进行分析。(C) ckap5敲除后不同时间段的细胞周期分析。在指定的时间点收获细胞，乙醇固定后用PI染色。(D) CKAP5敲低后细胞周期不同阶段细胞数量百分比的定量测量(E)处理48小时后对照细胞和CKAP5下调细胞纺锤体形成的共聚焦显微镜图像。图像以63× 7倍变焦拍摄。 Scale bars, 10 μm. (F) Quantitative analysis of the confocal microscopy images to determine the average number of bipolar spindle formation in control versus CKAP5 down-regulated cells. Graph shows % cells with indicated spindles in control and treated cells. Data are represented as average ± SEM of 50 events and analyzed by unpaired t test. ***P < 0.0001. (G) Quantitative comparison of the mitotic spindle length between control and treated cells. The distance between two mitotic poles was counted by the ImageJ analysis tool. Data represent average spindle length ± SEM of 40 events and analyzed by unpaired t test. ***P < 0.0001. (H) Reverse transcription PCR analysis of spindle checkpoint genes after 48 and 72 hours of treatment. Data represent average fold change compared to untreated samples from two representative experiments (n = 3). ns, not significant. Credit:科学的进步(2023)。DOI: 10.1126 / sciadv.ade4800
ckap5沉默NAR细胞的活细胞追踪。细胞用微管蛋白标记。GFP和组蛋白H2B。mCherry分别跟随有丝分裂纺锤体和细胞核。(A)实验示意图，显示LNP添加和活细胞成像的时间轴。(B)用siccontrol或siCKAP5-LNP处理的NAR细胞的细胞周期的代表性图像。用微管蛋白标记的NAR细胞。GFP和组蛋白H2B。mCherry分别用siccontrol或siCKAP5-LNPs (0.25 μg/ml)处理。处理后12小时用旋转盘共聚焦显微镜进行活细胞成像。每15分钟采集一次图像，持续48小时。比例尺，5 μm。 (C) Average time spent in mitosis in different groups as observed with a spinning disk microscope. Data are represented as average time ± SEM from 20 cell cycle events and analyzed by unpaired t test. **P = 0.0019. (D) Cell fate with respect to time in control and treatment groups. Each bar represents a cell and its fate through different phases of the cell cycle. Credit:科学的进步(2023)。DOI: 10.1126 / sciadv.ade4800
CKAP5沉默介导的对有丝分裂过程中NAR细胞微管动力学和定位的影响。(A)对照组和ckap5沉默组EB3定位的代表性表型。短暂转染EB3细胞。eGFP质粒标记微管+端。转染12小时后，细胞进一步转染siccontrol /siCKAP5 LNPs，并在sirna介导的沉默30小时后进行活细胞成像。活细胞成像采用超分辨率旋转圆盘显微镜。图像以100倍分辨率拍摄。比例尺，5 μm。在90秒的时间内，每秒捕获图像。图像表示91帧的叠加图像，每帧以特定的阴影标记，如时间帧标度所示。非动态+端由于其静态特性在所有90帧中相互叠加，由于叠加而表现为白色，而动态+端由于其动态特性而在所有90帧中不叠加，因此呈现为彩虹色。 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. Credit:科学的进步(2023)。DOI: 10.1126 / sciadv.ade4800
