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 .avi format. The imaging targets murine primary neurons using enhanced green fluorescent protein (EGFP).

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.

Methods

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 Cre-delivering
lentiviral particles (LVPs) or control LVPs and Pten-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 (>8 mW; 20 %) detecting EGFP and a
laser exciting at 561 nm (>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.