The astrocytes used in our study are primary cells isolated from the human brain cerebral cortex (#1800; ScienCell Research Laboratories). The culture method followed the manufacturer’s protocol. GlutaMAX media (10569010; Gibco) with 1% N-2 supplement (17502048; Gibco), 10% fetal bovine serum (26140079; Gibco), and 1% antibiotic (30-002-CI; Corning) was added and 80% confluence cells were cultured in a 100-mm cell culture dish (20100; SPL Life Sciences). Cells were incubated in 5% CO₂ at 37 ℃ conditions.

To evaluate the cytotoxicity of IS, we performed a thiazolyl blue tetrazolium bromide (MTT) assay. We seeded 5 × 10³ cells per well in a 96-well plate and incubated them for 24 hours. The IS was diluted and processed by concentrating in in a complete media (N-2 supplement with fetal bovine serum). After 7 days, the culture medium was removed, and 100 μL of MTT (M2128; Sigma-Aldrich) at a concentration of 0.5 mg/mL was added per well and incubated for 4 hours. Then, we removed the supernatant and add 100 μL of dimethyl sulfoxide to completely dissolve the generated formazan. Absorbance was measured at 570 nm using a plate reader (Multiskan Go; Thermo Fisher Scientific).

We used annexin V propidium iodide kit (556547; BD Pharmingen) to evaluate apoptosis/necrosis. For confocal imaging, cells were seeded on a sterilized cover glass coated with 2 μg/cm² poly-L-lysine solution (P4707; Sigma-Aldrich). After 24 hours of incubation, the IS was treated by diluting each concentration. Seven days later, annexin V (Alexa 488-conjugated) and propidium iodide (red-fluorescent) were diluted in the binding buffer provided at 1:100, treated and incubated at 37 ℃ in the dark for 15 minutes. After that, the staining solution was removed, and it was fixed with 4% paraformaldehyde for 5 minutes. Then, it was washed with phosphate buffered saline (PBS) and mounted with FluoroShield with DAPI (F6057; Sigma-Aldrich). The stained apoptosis/necrosis cells were also imaged with a confocal microscope (LSM710; Carl Zeiss).

Before fluorescence-activated cell sorting (FACS) analysis, cells were trypsinized and immuno-labeled with annexin V and propidium iodide. The cells were incubated in a buffer with fluorescent dyes under the manufacturer’s instructions. Then, they were analyzed using a FACSCalibur flow cytometer (BD FACSCanto II; BD Biosciences). The cells with fluorescein isothiocyanate (FITC) annexin V positive and propidium iodide negative were considered to undergo apoptosis.

To evaluate the effect of IS on the glycolytic function of astrocytes, we used a direct analysis of the concentration of dissociated hydrogen ions by measuring the extracellular acidification rate (ECAR) with an Agilent Seahorse XFe/XF analyzer. The XF Glycolysis Stress Test kit (103020-100; Agilent Technologies, Inc.) was used, and all experimental procedures followed the manufacturer’s user guide. The detailed methods are those referred to in our previous study [17,18]. Shortly, cells were seeded at 5 × 10³ cells per well in the plate included with the kit (102601-100, FluxPak mini for XF96 & Xfe96 Analyzers; Agilent Technologies, Inc.), and treated with IS by concentration. After 4 or 7 days, cells were washed twice with PBS, and the culture medium was replaced with XF DMEM Base Medium (103575-100; Agilent Technologies, Inc.), and incubated for 1 hour in a non-CO₂ incubator. After that, glucose (final concentration, 10 mM), oligomycin (final concentration, 1 μM), and 2-deoxyglucose (2-DG; final concentration, 50 mM) were added in cartridge ports A, B, and C, respectively. Then, the hydrated cartridge sensor was installed to the cell plate and the assay was performed.

To quantify intracellular proteins, we used western blotting. Astrocytes were seeded 5 × 10⁴ per well into a 12-well plate and incubated at 37 °C and for 24 hours. After that, IS was treated for each specified concentration and time. Then, the trypsinized cell pellet was lysed with radioimmunoprecipitation assay (RIPA) lysis and extraction buffer (89900; Thermo Fisher Scientific) on ice for 30 minutes. After centrifuging at 11,000 rpm for 5 minutes, the supernatant was used for immunoblotting. For protein quantification, Bradford protein assay (5000006; Bio-Rad Laboratories) was used according to the manufacturer’s instructions. Then, 30 μg of quantified protein was separated by size with an electrophoresis system (1658004; Bio-Rad Laboratories) using 10% gel (4561035; Bio-Rad Laboratories). This was then transferred to a polyvinylidene fluoride membrane (1620174; Bio-Rad Laboratories) and allowed to react in blocking buffer (5% skim milk in TBST) for 1 hour. Primary antibodies were added and incubated overnight in a cold room. We purchased antibodies for hexokinase 1 (HK1), pyruvate kinase isozyme M2 (PKM2), pyruvate dehydrogenase (PDH), phosphofructokinase (PFKP), and lactate dehydrogenase (LDH), which were included in the cell signaling glycolysis sampler kit (8337T; Cell Signaling Technology). Antibodies for Bcl2 (4223T; Cell Signaling Technology), Bax (89477S; Cell Signaling Technology) and β-actin (3700S; Cell Signaling Technology) were used. All processes were followed manufacturer’s instructions. After washing three times with TBST for 5 minutes each, the HRP-linked secondary antibodies were diluted 1:2,000 and allowed to react for 30 minutes. They then reacted with the enhanced chemiluminescence solution (RPN2106; GE healthcare) and were detected with an imaging system (FUSION Solo S; Vilber). Each membrane was compared with β-actin as a control, and ImageJ software was used for quantitative analysis.

We imaged morphological changes of astrocytes with a three-dimensional (3D) cell imaging machine (3D Explore; Nanolive) as in our previous studies [17,18]. Briefly, astrocytes were seeded into a FluoroDish (FD35-100; World Precision Instruments) and incubated. Except for the IS treatment, after 24 hours, the astrocytes were treated with IS for 7 days, and those in a control group were incubated under the same conditions. Then, 3D images of astrocytes from both groups were acquired.

We analyzed the morphological changes of cytotoxicity following IS treatment (at 7 days) in human astrocytes using a 3D cell analyzer. In the IS-treated astrocytes, we observed such apoptotic cell morphological changes as blebbing and shrinkage of the cytosolic area as compared to the control group (Fig. 1).

We performed an MTT assay to evaluate the viability of astrocytes as a marker of cytotoxicity by IS. As shown in Fig. 2, after 4 and 7 days, IS reduced astrocyte cell viability in a dose-dependent manner. In order to investigate whether this decreased cell viability was induced by apoptosis as shown in Fig. 1, we did a FACS study. After incubation with IS for 4 and 7 days, it was noted that the proportion of apoptotic cells had significantly increased (Fig. 3). Apoptotic cells were more prominent after 7 days of IS incubation as compared to those after 4 days. This effect was also more noticeable at higher IS dose. An immunoblot study confirmed that the Bax/Bcl-2 ratio had significantly increased by days 4 and 7 in a dose-dependent manner (Fig. 4A, B). Astrocytes incubated both with or without IS (0.5 and 1.0 mM) for 7 days were stained with annexin V, FITC (green), and propidium iodide (red). As shown in Fig. 4C, astrocytes without IS were not stained by either annexin V or propidium iodide. However, cells treated with 0.5 mM of IS were partly stained by either of these and those treated with 1.0 mM were significantly stained. Thus, we were able to readily observe co-staining in these spots.

We investigated whether IS could affect glycolytic activity in human astrocytes. ECARs were investigated as surrogates of glycolysis by quantifying lactate production. The ECAR was measured during the sequential addition of glucose (the substrate of glycolysis), oligomycin (a selective inhibitor of mitochondrial respiration), and 2-DG (a specific inhibitor of glycolysis). IS significantly suppressed ECAR levels in response to glucose as compared to controls on days 4 and 7, and the degree of suppression was dose-dependent (Fig. 5). Decreased adenosine triphosphate (ATP) levels were also observed.

Next, we investigated whether IS could regulate enzyme expression in the glycolysis pathway. On day 4, IS decreased the PDH and PFKP protein levels as compared to the control, but the PKM2 seemed to increase slightly (Fig. 6A, B). After 7 days of IS treatment, HK1, PKM2, PDH, and PFKP protein levels were decreased as compared to the control (Fig. 6C, D). These results suggest that glycolysis inhibition in human astrocytes is associated with the exposure time and dose of IS.