Time lapse imaging analysis of the effect of ER stress modulators on apoptotic cell assessed by caspase3/7 activation in NG108-15 cells

This paper reports the data from the long term time lapse imaging of neuronal cell line NG108-15 that were treated with apoptosis inducer or various ER stress inducers. Use of the fluorescent reporter for activated caspase3/7 in combination with the conventional light microscope allowed us to investigate the time course of apoptosis induction at the single cell level. Quantitative as well as qualitative data are presented here to show the effect of two different ER stress modulating chemical compounds on caspase3/7-dependent apoptosis in neuronal cell line NG108-15 cells. Additional results and interpretation of our data concerning ER stress and apoptosis in NG108-15 cells can be found in Suga et al. (2015) [1] and in Suga et al. (2015) [2].

Type of data text file, graph, figure How data was acquired

Microscope, time lapse imaging, luminometer
Data format analyzed Experimental factors NG108-15 cells were treated with apoptosis inducer or different ER stress inducer together with chemical compounds that modulated ER stress and viability assays. Experimental features ER stress or apoptosis induced cells were processed for time lapse imaging

Data source location
Kyorin University School of Medicine, Tokyo, Japan Data accessibility All data are provided in this article

Value of the data
The results provided here may inform many researchers that are investigating the relationship of ER stress, apoptosis, and neuronal cell death.
The data provides important information on the use of various ER stress inducers and modulators in neuronal cell lines.
Data of the time course and the population of the cells showing caspase3/7 dependent apoptosis presented here can serve as a benchmark to the researchers who investigate the effect of ER stress using end-point assays.

Data
We recently showed that syntaxin5 (Syx5) protein, one of the ER-Golgi SNARE proteins, is upregulated by ER stress, but downregulated by caspase3-dependent apoptosis in neuronal cells [1][2][3][4]. Importantly, caspase3 has been identified as a key mediator of neuronal cell death [5], and it has been implicated that caspase3 is a potential target for pharmacological therapy during early stages of  Alzheimer's disease [5]. We showed that sustained ER stress promotes caspase3-dependent apoptosis during the later phase of the ER stress response in NG108-15 cells [1,2]. In addition, we and others have previously shown that caspase3-mediated cleavage of Syx5 protein accompanies inhibition of secretory traffic during apoptosis [2,6]. Here, we used a long term real time imaging technique to analyze the effect of various toxins and reagents that affect ER stress on the caspase3/7-dependent apoptosis of neuronal cell line NG108-15 cells.

Experimental design, materials and methods
We first examined the cell viability of NG108-15 cells treated with the strong apoptosis inducer Staurosporine (STS) and various toxins that cause ER stress (Fig. 1). Apoptotic cells were defined by the activation of the caspase3/7 fluorescent reporter, which was assessed by the appearance of green fluorescence emission in the cells. Induction of apoptosis by STS caused prominent activation of caspase3/7, whereas vehicle treated control cells show no fluorescence after 24 h of treatment (Fig. 3). We utilized two different ER stress modulators (Fig. 2). One is Salubrinal (Salub) that has been shown to protect cell from ER stress [7], and the other is a chemical chaperone sodium 4-phenylbutyrate (PBA) that has been reported to rescue the proteolytic deficit [8]. We first examined the effects of ER stress modulating reagents Salub and PBA on ER stress-induced apoptosis using time lapse imaging (Fig. 2). We examined their effects on STS-induced apoptosis in NG108-15 cells and showed the different effects of these two modulators (Fig. 3). In order to see their effects on ER stress-induced apoptosis triggered by ER stress, we used three different types of toxins for the ER stressors. Treatment of NG108-15 cells with Thapsigargin (Tg), a potent ER stress-inducing toxin that perturbs ER Ca 2 þ homeostasis by inhibiting sarco/endoplasmic reticulum Ca 2 þ -ATPase, caused gradual increase in apoptotic cells after 24 h of treatment (Fig. 4). We also treated cells with the fungal toxin Brefeldin A (BFA) (Fig. 5), which is known to induce accumulation of proteins in the ER due to inhibition of protein trafficking through the early secretory compartments [9]. Cells were also treated with Tunicamycin (Tm), a toxin that inhibits N-linked glycosylation of proteins in the ER (Fig. 6). Data showing the difference in the action between Salub and PBA on the alleviation of the caspase3/7 dependent apoptosis induced by various toxins and reagents are presented (Figs. 3-6).

Materials
Tm and BFA were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). Salb was purchased from Enzo (Farmingdale, NY). STS, Tg, and PBA were purchased from Merck (Darmstadt, Germany). All other reagents were of the highest grade available, unless otherwise noted.

Cell culture
Mouse neuroblastoma and rat glioma hybrid NG108-15 cells were cultured in Dulbecco's modified Eagle's medium containing 4 mM L-glutamine, 100 U/mL of penicillin, 100 mg/mL of streptomycin, Although the ER stress-triggered cell death is not only due to caspase3/7, but compared to the ratio of dead cells obtained from the cell viability assay of STS-treated cells (Fig. 1), it can be estimated that approximately 79% of dead cells are caspase3/7 positive after 16 h of treatment. PBA, but not Salub, significantly alleviated STS-induced caspase3/7 dependent apoptosis. Scale bar, 50 μm.

Real-time imaging of caspase 3/7 activation
NG108-15 cells were plated in glass-bottomed 35-mm dishes coated with PLL (Sigma-Aldrich) prior to the day of imaging. Cells were incubated in culture medium containing CellEvent caspase3/7 reagent (Life Technologies, Rockland, MD, USA) for 30 min in a humidified chamber maintained at 5% CO 2 and 37°C. After incubation, 2 mL of fresh medium was added and the dish was placed in an imaging chamber kept under the same conditions as the CO 2 incubator using a stage-top chamber equipped with a digital gas mixer (GM-8000, TokaiHit, Shizuoka, Japan). In the early frames at the beginning of real time imaging, rounding of the cell is observed due to the stimuli of changing to the fresh medium done just before the starting of time lapse imaging. The rounding of cells during the cell cycle can be also observed before cell division. Digital images of stained cells were acquired using an IX81 inverted microscope (Olympus) equipped with a 20 Â UPLSApo lens (numerical aperture, 0.75) and a CoolSNAP-HQ CCD camera (Roper Scientific) using appropriate filters for emission. LED (CoolLED) was used for excitation. Images were acquired every 10 min and collected as TIFF files using MetaMorph software (Universal Imaging). Whole number of cells and caspase3/7 positive cells were counted within the area of the acquired image (450 Â 335 mm 2 ) from each time point. Apoptotic cells were defined by the activation of the caspase3/7 fluorescent reporter, which was assessed by the

Cell viability assay
Cell viability was measured with a CellTiter-Glo Luminescent Cell Viability Assay system (Promega) according to the manufacturer's instructions. Briefly, cells were harvested in PLL-coated 96well plates (Greiner Bio-One) and treated with various reagents. Subsequently, the intracellular ATP levels were determined by a bioluminescence reaction using thermostable recombinant firefly luciferase and beetle luciferin. The luminescence was measured using a GloMax Luminometer (Promega). The level of luminescence produced is proportional to the cell number present in each well.

Data analysis and statistics
Data are presented as mean 7 SEM where otherwise noted, with 'n' indicating the number of samples examined. For the cell viability analysis, a t-test was used to determine the statistical significance of differences between values. Data sets from time-lapse imaging were subjected to oneway ANOVA and Bonferroni multiple comparisons analysis with post-hoc tests. *P o0.05.
Supplementary data associated with this article can be found in the online version at http://dx.doi. org/10.1016/j.dib.2015.11.030.