Inventory of Supplemental Information

Inventory of Supplemental Information

1. Supplementary Results

C6GSLCs have the properties of neural stem cells, with elevated expression of CD133.

EGCG inhibited C6GSLC growth more potently than TMZ.

EGCG effectively inhibited the stem-like characteristics of C6GSLCs.

EGCGsynergized with TMZ and was associated with P-gpinhibition in C6GSLCs.

2. Supplementary Figures

 Supplementary Fig.1.C6GSLCs have the properties of neural stem cells in neurosphere culture.

 Supplementary Fig.2.EGCG inhibited C6GSLC growth more potently than TMZ.

 Supplementary Fig.3.EGCGaffectedcell viability,neurosphere formation, and migration in C6GSLCs.

 Supplementary Fig.4.EGCG in combination with TMZ significantly reduced cellviabilityinC6GSLCs.

 Supplementary Fig.5.EGCGsynergized with TMZ, associated with P-gpinhibition in C6GSLCs.

Supplemental Information

EGCG Inhibits Properties of Glioma Stem-like Cells and Synergizes with Temozolomide through Downregulation of P-glycoprotein Inhibition

Yong Zhang • Shao-Xiang Wang • Ji-Wei Ma • Hai-Ying Li • Jie-Cheng Ye • Si-MingXie • Bin Du • Xue-Yun Zhong

Supplementary Results

C6GSLCs have neural stem cell properties and highly express CD133

C6GSLCs were obtained by neurosphere culture anddeveloped neural stem cell properties. The cancer stem cell marker CD133 was significantly increased, whereas the expression of the astrocyte differentiation marker GFAPwas decreased in C6GSLCs (supplementary Fig. 1). These data indicate that C6GSLCs have characteristics of neural stem cells in terms of elevated CD133 expression.

EGCG inhibited C6GSLC cell growth more potently than TMZ

To investigate the inhibitory effect of EGCG and TMZ on cell growth in C6GSLCs, we examined cell viability following EGCG and TMZ treatment using a CCK-8 assay. As shown in supplementary Fig. 2,TMZIC50 values in C6 and C6GSLCs were 526.36 μM and 1089.01 μM, respectively; the EGCG values were 56.71 μM and 181.23 μM, respectively.These data indicated that EGCG may be a useful agent to target C6GSLCs.

EGCG treatment effectively inhibited the stem-like characteristics of C6GSLCs

To investigate whether EGCG inhibited the stem-like characteristics of C6GSLCs effectively,C6GSLCs were dissociated in suspension and treated with EGCG (0-200 μM). After EGCG treatment, we observed morphology changes and a reduction in cell viability in C6GSLCs (supplementary Fig. 3). In addition,EGCG reduced sphere-forming efficiency and migration of C6GSLCs in a dose-dependent manner.These results suggest that the stem-like characteristics of C6GSLCs can be effectively inhibited by EGCG.

EGCG synergized with TMZ was associated with P-gp inhibition in C6GSLCs

C6GSLCs were treated with vehicle, 100 μMEGCG, 100 μMTMZ, 50 μM Verapamil (VER),EGCG combined with TMZ, or VER combined with TMZ for 48 h. Cell viability was examined in C6GSLCs by CCK-8 assay. Asshown in supplementary Fig. 4,EGCG in combination with TMZ significantly reduced the viability of C6GSLCs.

Furthermore, we measured P-gp expression and apoptosis by immunofluorescence staining after treatment. Expression of P-gp was decreased compared to the control group, and apoptotic cells were detected by TUNEL assay in the EGCG treatment group and the Verapamil (VER) treatment group. There were no differences in P-gp expression or apoptosis between the TMZ treatment group and the control group. It obviously reduced the expression of P-gp and more obviously the apoptotic cells were detected in the TMZ + EGCG treatment group and VER + EGCG treatment group. Moreover, the expression of P-gp was drastically downregulated in the apoptotic cells indicated by white arrows (Supplementary Fig. 5). These results suggest that EGCG may synergize with TMZ to inhibit P-gp in C6GSLCs.

Supplementary Figure Legends

Supplementary Fig. 1.C6GSLCs have properties of neural stem cells in neurosphere culture. The glioma cell line C6 was able to continually divide and form neurospheres after 7 days in serum-free DMEM/F12 supplemented with B-27,EGF, and bFGF.mRNA expression of CD133,Nestin, and GFAP were measured by RT-PCR and normalized against GAPDH in C6 and C6GSLCs.The specific PCR primer sequences for these genes, designed by Primer Premier 5.0 software, were as follows: CD133 forward: 5'- CTGCCCAGAGTGGAAGAA-3'; CD133 reverse: 5'-CACAGCAAGCCCAGGTAA-3'; Nestin forward: 5'-GCAGGCCACTGATAAGTTCCA-3'; Nestin reverse: 5'-CCTCCAGCAGAGTCCTGTATGT-3'; GFAP forward: 5'-GAGTGGTATCGGTCCAAGTT-3'; GFAP reverse: 5'-CTCAAGGTCGCAGGTCAA-3'; GAPDHforward:5'-CGGCAAGTTCAACGGCACAG-3'; GAPDH reverse: 5'-CGCCAGTAGACTCCACGACAT-3'. Data represent the mean ± SD. *, # = significantly different from the respective controls, *,P < 0.05, #, P < 0.01.

Supplementary Fig. 2.EGCG inhibited C6GSLC cell growth more potently than TMZ.C6GSLCs were dissociated with TrypLE Express reagent (Invitrogen, Carlsbad, CA) and filtered through a 40-μm cell strainer.Cell viability was measured using a CCK-8 assay after treating C6 and C6GSLCs for 48 h with various doses of TMZ (A) and EGCG (B).

Supplementary Fig. 3.EGCG affected cell viability,neurosphere formation, and migration in C6GSLCs.A,C6GSLCs were dissociated with TrypLE Express reagent and treated with EGCG (0-100 μM) for 7 days in 96-well plates.Scale bars: 50 μm.B, Cell viability was measured using trypan blue, and live cells were counted under a microscope in five random fields per well.C,C6GSLCs were seeded in suspension after dissociation and treated with EGCG (0-200 μM) for 48 h. Cells were then cultured for 7 days in serum-free DMEM/F12 supplemented with B-27,EGF, and bFGF to check sphere-forming efficiency. The number of C6GSLCs was counted under a microscope in five random fields per well.D, Transwell migration assay.C6GSLCs were plated at a density of 5 × 104 cells/ml in the top chamber of a transwell and treated with EGCG (0-200 μM) for 24 h. Cells that migrated to the lower chambers were fixed with methanol and stained with crystal violet. Cells were counted under a microscope in five random fields per well. Data represent the mean ± SD. * = different from the respective controls,P < 0.05.Scale bars: 100 μm.

Supplementary Fig. 4.EGCG in combination with TMZ significantly suppressed the viability of C6GSLCs.C6GSLCs were seeded in suspension and treated with vehicle, 100 μMEGCG, 100 μMTMZ, 50 μM Verapamil (VER),EGCG + TMZ, or VER + TMZ for 48 h.A, The morphology changes in C6GSLCs were observed with light microscopy after treatment.B,We examined the viability of C6GSLCs by CCK-8 assay.Data represent the mean ± SD. * = different from the respective controls, #, & = significant difference between the two connected objects,Kruskal-Wallis test,P < 0.05.Scale bars: 100 μm.

Supplementary Fig. 5.EGCG synergized with TMZ, which was associated with P-gp inhibition in C6GSLCs.Confocal immunofluorescence staining.C6GSLCs were seeded in suspension and treated with vehicle, 100 μMEGCG, 100 μMTMZ, 100 μMVER,EGCG + TMZ, or VER + TMZ for 48 h.Blue fluorescence represents DAPI; red fluorescence represents P-gp; green fluorescence represents TUNEL.Apoptotic cells are indicated by white arrows. Scale bars: 50 μm.

Supplementary Fig.1

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Supplementary Fig.5