Supplementary MaterialsAdditional file 1: Table S1. Additional file 6: Figure S2. Stemness properties of LGR5+ 8591 cells in vitro. (a) Cell proliferation assays of LGR5+ and LGR5??8591 cells (test). Scale bar?=?100?m. (c) Drug resistance curve of TMZ in LGR5+ and LGR5??8591 cells. (d) Images and numbers of invasive cells in invasion assays (top, test) and images and numbers of migrated cells in migration assays (bottom, test). All data are represented as mean??SD from triplicate wells. ***, test). Error bars represent the mean??SD. (e) The correlation between the levels of LGR5 and Ki67 by Spearman correlation analysis (test was used to analyze the differences in the results between groups. Comparisons among three or more groups were assessed using a one-way analysis of variance (ANOVA). Comparison between two or more groups in different time points were TLR9 assessed by two-way ANOVA. Correlations between LGR5 and Ki67, N-cadherin, SOX2 and CD44 expressions were analyzed by Spearman correlation method. All values are expressed as means SD. The correlation between LGR5 expression and clinicopathological variables was analyzed by a 2 test or Fishers exact test. OS and PFS curves were plotted by the Kaplan-Meier method and compared using the results of a Log-rank test. The Cox proportional hazards model was used to estimate the independent prognostic factors for OS and PFS in the multivariate analysis. values less than 0.05 were considered statistically significant. Results Percentage of LGR5+ cells is higher in enriched cells than that in parent cells To determine the expression and localization of LGR5 in glioma cells, LGR5 staining was performed in 3 types of glioma cell lines (U251, U87 and A172) and 3 types of human primary P7C3-A20 inhibition glioma cells (8591, LHH and 7112), demonstrating that LGR5 was expressed in the cell membrane and cytoplasm (Fig.?1a). All of the abovementioned glioma cells were proved to be derived from astrocytes by glial fibrillary acidic protein (GFAP) co-dyeing (Fig. ?(Fig.1a).1a). We obtained enriched cells from parent cells through serum-free enrichment, which is a method for quickly screening GSCs. The expression of LGR5 was detected in glioma parent cells and in enriched cells by FCM (Additional?file?5: Figure S1a). The positive proportions of LGR5 were 2.46%, 2.01%, 5.76%, 1.34%, 1.79% and 1.45% in U251, U87, A172, 8591, LHH and 7112 parent cells, respectively, and 21.50%, 11.23%, 16.04%, 15.42%, 11.41% and 4.53% in U251, U87, A172, 8591, LHH and 7112 enriched cells, respectively. The positive rates of LGR5 in enriched cells were 8.7, 5.6, 2.8, 11.5, 6.4 and 3.1 times higher than those in parent cells for U251, U87, A172, 8591, LHH and 7112, respectively. Thus, we chose U251 and 8591, P7C3-A20 inhibition whose enrichment levels were the highest (Fig. ?(Fig.1b),1b), to establish cell models. Then, LGR5+ and LGR5? cells were obtained by FACS to perform follow-up experiments P7C3-A20 inhibition (Additional file 5: Figure S1b). Open in a separate window Fig. 1 LGR5 expression in different glioma cells, and stemness properties of LGR5+ U251 P7C3-A20 inhibition cells in vitro. a The expression and localization of LGR5 and GFAP in the glioma cell lines (U251, A172 and U87MG) and the human primary glioma cells (8591, LHH and 7112). Scale bar?=?30?m. b The enrichment levels of LGR5 expression by FCM in parent cells and enrichment cells. c Cell proliferation assays of LGR5+ U251 cells and LGR5? U251 cells (test). Scale bar?=?100?m. e Drug resistance curve of TMZ in LGR5+ and LGR5? U251 cells. f Images and numbers of invasive cells in invasion assays (top, test) and images and numbers of migrated cells in migration assays (bottom, test). g Western blot analysis for LGR5 and N-cadherin in LGR5+ and LGR5? U251 cells. The expression levels of LGR5 and N-cadherin were quantified by Image lab software by densitometric analysis and were normalized to the control groups. Human -actin was used as the internal control. (test). All.