SHG44 human glioma cells from the American Type Culture Collection (ATCC, Manassas, VA, USA) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C in a humidified incubator with 5% CO₂. Tumor cells grown to 80% confluence were rinsed with sterile phosphate-buffered saline (PBS) (0.01 M, pH 7.4) and digested at 37°C with 0.25% trypsin for 1 min, then resuspended in a serum-free neural stem cell medium consisting of D-MEM/F-12, 2% B27 (Gibco BRL, Gaithersburg, MD, USA), 20 ng/ml EGF (PeproTech, Inc., Rocky Hill, NJ, USA), 20 ng/ml bFGF (PeproTech, Inc.), and then seeded into 6-well suspension cell culture plates (Beaver, China). After 7 days of culture at 37°C in 5% humidified CO₂ atmosphere, the medium was removed by centrifugation and GSC-forming spheres were digested with 0.25% trypsin into a single-cell suspension. GSCs were subcultured and enriched three times before being used for assays.

GSCs grown on polylysine-coated coverslips were fixed using 4% paraformaldehyde for 15 min and permeabilized with 0.5% Triton X-100 (PBST) for 10 min. The coverslips were blocked with 5% bovine serum albumin (BSA) in PBS (0.01 M, pH 7.4) for 30 min at room temperature, followed by incubation with primary antibodies targeted against rabbit anti-CD133 (#PAB12663; 1:200; Abnova), mouse anti-nestin (#ab18102; 1:200; Abcam) and rabbit anti-Bcl-2 (#ab32124; 1:100; Abcam) overnight at 4°C. After three washes with PBS, the coverslips were incubated with PE-labeled rabbit anti-mouse IgG (#ab7000; 1:100; Abcam) and FITC-labeled goat anti-rabbit IgG (#ab6717; 1:1,000; Abcam) for 120 min at 37°C. Nuclear DNA was labeled in blue with DAPI. The staining results were imaged on a Nikon C-1 laser-scanning confocal microscope (Nikon, Tokyo, Japan).

After the cells were grown to sub-confluence, they were supplemented with various concentrations (10, 20, 40, 80 and 160 µM, respectively) of ISL (purity ≥98%; Shanghai Yuanye Bio-Technology Corp., Shanghai, China), 2.5 µM DAPT (a Notch/γ-secretase inhibitor) (purity >98.5%; Gene Operation, USA) or 10% FBS (as positive control) for different times. Treatment effects on proliferation and differentiation were examined by further assays described below.

Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) assay was used to measure cell proliferation of the GSCs. Briefly, 3×10³ cells per well were seeded into 96-well suspension cell culture plates (Beaver) in triplicate. At the appropriate time (12, 24, 48 and 72 h), 10 µl CCK-8 solution was added to each well, and the plates were incubated for 4 h at 37°C under a moist atmosphere with 5% CO₂. The absorbance at 450 nm was measured using the Gen5 microplate reader (BioTek, Winooski, VT, USA).

The formation of GSC spheres was observed and imaged after 9 days of treatment (ISL was supplemented every other day) using an Olympus IX71 Inverted Microscope (Olympus, Tokyo, Japan). The diameters and numbers of GSC-forming spheres were calculated from 5 random fields of view by Image-Pro Plus 6.0 software.

After 48 h of treatment, GSCs grown on polylysine-coated coverslips were fixed in 4% paraformaldehyde for 15 min and permeabilized with 0.5% Triton X-100 (PBST) for 10 min. The coverslips were blocked with 5% BSA in PBS (0.01 M, pH 7.4) for 30 min at room temperature, followed by incubation with primary antibodies targeted against mouse anti-GFAP (#ab4648; 1:50; Abcam), mouse anti-β III Tubulin (#ab78078; 1:1,000; Abcam) and rabbit anti-Hes1 (#ab119776; 1:100; Abcam) overnight at 4°C. After three washes with PBS, the coverslips were incubated with PE-labeled rabbit anti-mouse IgG (#ab7000; 1:100; Abcam) and FITC-labeled goat anti-rabbit IgG (#ab6717; 1:1000; Abcam) for 120 min at 37°C. Nuclear DNA was labeled in blue with DAPI. The fluorescent sections were observed and photographed with a Nikon C-1 laser-scanning confocal microscope (Nikon). The number of GFAP⁺ cells and β III tubulin⁺ cells were determined in 5 random fields of view by Image-Pro Plus 6.0.

The cultured cells were harvested and lysed at 48 h after ISL and DAPT treatment. Total cellular RNA was extracted using TRIzol reagent (Takara Biotechnology Co., Ltd., Dalian, China). To remove any DNA contamination, RNA samples were treated with Recombinant DNase I (Takara Biotechnology). Single-stranded cDNA was synthesized from total RNA with the PrimeScript™ RT Master Mix (Takara Biotechnology). qPCR was performed for HES1 and internal control GAPDH using the SYBR Premix Ex Taq™ II (Takara, Shiga, Japan) on a 7300 real-time PCR system (Applied Biosystems, Singapore, Singapore). All reactions were carried out in triplicate and expression data (after being calibrated with GAPDH levels) were analyzed using the 2^−∆∆Cq method. Optimal oligonucleotide primers used in the above PCR assays were designed and synthesized by Takara Biotechnology based on published human cDNA sequences. The primer sequences were as follows: HES1 sense 5′-GTGTCAACACGACACCGGATAAAC-3′ and antisense 5′-CAGAATGTCCGCCTTCTCCAG-3′; GAPDH sense 5′-GCACCGTCAAGGCTGAGAAC-3′ and antisense 5′-TGGTGAAGACGCCAGTGGA-3′. All reactions were run in triplicate.

The protein expression levels of differentiation markers (GFAP and β III tubulin) and Notch1 signaling pathway markers (Notch1 and Hes1) were examined by western blotting. β-actin levels were evaluated as a loading control. Briefly, cells were lysed and homogenized in RIPA lysis buffer (Solarbio, Beijing, China). The supernatants were obtained by centrifugation at 4°C for 10 min at 12,000 rpm and the total protein contents were quantified using a BCA kit (Solarbio). For electrophoresis, equal amounts of protein (15 µg/lane) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis on 8, 10 or 12% polyacrylamide gel depending on the target protein and transferred onto PVDF membranes (Millipore, Billerica, MA, USA) using a wet electroblotting system (Mini Trans-Blot; Bio-Rad). For immunoblotting, the membranes were blocked in 5% non-fat dry milk for 2 h at room temperature and incubated overnight at 4°C with one of the following primary antibodies: GFAP (#ab4648; 1:400; Abcam), β III tubulin (#ab78078; 1:800; Abcam), Notch1 (#ab128076; 1:800; Abcam), Hes1 (#ab119776; 1:800; Abcam) and β-actin (#AP0060; 1:3,000; Bioworld Technology Inc.). After three washes in Tris-buffered saline containing 0.5% Tween-20 (TBST), the samples were treated with HRP-conjugated goat anti-rabbit (#E030120-01; 1:10,000; EarthOx Life Science, Millbrae, CA, USA) or goat anti-mouse secondary antibodies (#ab6728; 1:10,000; Abcam) for 1 h at room temperature. The antibody labeling was visualized using enhanced chemiluminescence reagent (Millipore) and exposed on X-ray film (Kodak, China). For the immunoblot analyses, densitometry was performed with Image-Pro Plus 6.0 software. The optical densities (ODs) of the protein bands were calculated relative to the ODs of the reference protein β-actin.

Data are expressed as the mean ± SD. All statistical analyses were performed using the SPSS software program (version 21.0; IBM Corp., Armonk, NY, USA). The differences between the experimental groups were analyzed by one-way analysis of variance (ANOVA). P<0.05 was considered statistically significant.

Within 48–72 h of serum-free culture SHG44 human glioma cells yielded a minority fraction of cells that possessed extensive proliferative and self-renewal potential, and generated free-floating neurosphere-like brain tumor spheres. These spheres gradually enlarged as the time of the culture increased (Fig. 1B and C). In comparison, glioma cells cultured in DMEM with 10% FBS growed adherently (Fig. 1A).

Expression of GSC surface markers were examined in human GSC-forming spheres. As shown in Fig. 1D, the cells expressed characteristic neural stem cell (NSC) markers CD133 and Nestin. SHG44 human glioma cells and tumor neurospheres were postive for the proto-oncogene Bcl-2 which distinguished them from normal neurons and NSCs (Fig. 1E and F). The immunofluorescence staining data indicated that the tumor spheres expressed typical surface markers of GSCs and thus were used for experiments as described below.

To investigate the influence of ISL on GSC proliferation, we firstly examined the activities of cells treated with various concentrations of ISL for different times. As shown in Fig. 2A, within 24 and 48 h of treatment the proliferative activity of the cells was significantly higher in the 10, 20 and 40 µM ISL groups than that noted in the 0 µM ISL group. There was no significant difference between the 0 and 80 µM ISL group within 24 h, but the activity of cells began to decline after 48 h. After 72 h, the cell proliferation of the ISL treatment groups were obviously inhibited in a dose-dependent manner, with a half inhibitory concentration IC₅₀ of 102.744 µmol/l.

Formation of neurospheres is a morphological feature of GSCs. Then we examined the influence of ISL on GSC-forming spheres on the 9th day of treatment. It was found that the 0 µM ISL group had the largest diameter and highest number of tumor spheres formed from the GSCs (Fig. 2B-D). The diameters and the numbers were both decreased by ISL in a dose-dependent manner, and 160 µM ISL completely prevented neurophere formation, displaying a similar tendency with cell activity at 72 h.

To determine whether ISL promotes the differentiation of GSCs, intracellular differentiation markers GFAP and β III tubulin were examined after 48 h of ISL treatment by immunofluorescence staining. As shown in Fig. 3, the numbers of differentiation marker-positive cells were higher in the ISL group and DAPT group than that in the control group after 48 h. As a comparison, FBS effectively induced the differentiation of GSCs.

To determine whether ISL inhibits Hes1 (Notch1 target gene) in the differentiated cells, GFAP and Hes1 were assessed together after the cells were treated for 48 h. The relative intensity of Hes1 was decreased in the GFAP⁺ cells induced by ISL (Fig. 4).

The protein expression levels of differentiation markers were also examined after 48 h of ISL treatment. As expected, the protein expression levels of GFAP and β III tubulin in the DAPT group and ISL groups were higher than those of the control, and DAPT induced higher GFAP and β III tubulin protein expression levels compared to the ISL groups. However, there was no obvious difference between the low dose (20 µM) and high dose (80 µM) ISL groups (Fig. 5A and B).

To examine whether ISL inhibits the proliferation and induces the differentiation of human GSCs through Notch1 signaling, Notch1 and Hes1 were analyzed 48 h after GSCs were respectively treated with ISL and DAPT. The predicted molecular weight of Notch1 is 272 kDa. In the present study, the band observed at 120 kDa could potentially be a truncated form of Notch1, following cleavage by furin-like protease. The protein expression levels of Notch1 in the DAPT group and ISL groups were lower than those of the control (Fig. 5C). The mRNA and protein expression levels of Hes1 were also significantly decreased compared to the control (Fig. 5D and E). DAPT induced lower Notch1 and Hes1 expression levels compared to the ISL groups.