Data Set 1: Live cell microscopy videos of cellular distribution of PTEN:EGFP and PTEN-L:EGFP after 50µM glutamate stress in mouse primary neurons (Fig. 3a-b)

This dataset contains 8 live cell microscopy timelapse video files in <b>.avi</b> format. The imaging targets murine primary neurons using enhanced green fluorescent protein (EGFP).<br>The related study investigates the function and regulation of PTEN-L in neurons in the context of brain ischemia. Exogenous, EGFP-tagged PTEN-L or the shorter variant PTEN were expressed in primary neurons after knock out of endogenous PTEN. Oxygen-glucose deprivation or exposure to 50 µM glutamate were applied to neurons to test their response to ischemic-like stress.<br><b>Methods</b><br>Cells expressing PTEN:EGFP or PTEN-L:EGFP were imaged every 10 min for 90 min. The time-lapse movies compiled from the 90 min observation. Primary neurons derived from conditional PTEN knockout mice were transduced with both <i>Cre</i>-delivering lentiviral particles (LVPs) or control LVPs and <i>Pten</i>-delivering LVPs or control LVPs as described above and seeded in 8-well microscopy dishes (Ibidi). DIV 9 primary neurons were imaged while maintaining culturing conditions (20 % oxygen, 5 % CO₂ and 37°C). A confocal microscope (Nikon Ti2) with uniform spinning disk illumination (Andor Borealis), an EMCCD Camera (iXon3 DU-888 Ultra) and 60x Plan Apo oil objective (Nikon) was used to acquire images. 8 positions across 4 wells were selected and images of z-planes (30 - 40 µM with 1 µM intervals) were taken with a laser exciting at 488 nm (&gt;8 mW; 20 %) detecting EGFP and a laser exciting at 561 nm (&gt;15 mW; 9 %) detecting mCherry. Next, cells were treated with 50 µM glutamate and each position was imaged every 10 min for 90 min using above described settings. Fiji software was used to compile time lapse videos over the 90 min observation period. <br>

本数据集包含8个.avi格式的活细胞显微延时视频文件。成像对象为经增强绿色荧光蛋白（enhanced green fluorescent protein, EGFP）标记的小鼠原代神经元。
相关研究围绕脑缺血场景下神经元中PTEN-L的功能与调控机制展开。实验中先敲除内源性PTEN，随后向原代神经元中外源表达EGFP标记的PTEN-L及其截短变体PTEN。通过氧糖剥夺或暴露于50 μM谷氨酸，以检测神经元对类缺血应激的应答反应。
**实验方法**
对表达PTEN:EGFP或PTEN-L:EGFP的细胞每10分钟进行一次成像，持续90分钟，最终将观测数据整合为90分钟的延时视频。本研究使用的原代神经元来自条件性PTEN敲除小鼠，按照前述方法，分别用携带Cre重组酶的慢病毒颗粒（LVPs）、对照慢病毒颗粒，以及携带Pten的慢病毒颗粒、对照慢病毒颗粒进行转导，随后接种于8孔显微成像培养皿（Ibidi）。在维持标准培养条件（20%氧气、5% CO₂、37℃）的情况下，对体外培养第9天（DIV 9）的原代神经元进行成像。
成像采用共聚焦显微镜（Nikon Ti2），搭配均匀旋转盘照明系统（Andor Borealis）、EMCCD相机（iXon3 DU-888 Ultra）及60× Plan Apo油浸物镜（Nikon）。选取4个培养孔中的8个成像位点，采集z轴层面图像（层间距1 μM，总厚度30~40 μM）：使用488 nm激光（功率>8 mW，占比20%）激发并检测EGFP信号，使用561 nm激光（功率>15 mW，占比9%）激发并检测mCherry信号。
随后向培养基中加入50 μM谷氨酸，按照前述参数每10分钟对每个位点成像一次，持续90分钟。使用Fiji软件将90分钟观测周期内的图像整合为延时视频。