Figure 3. SPP1 expression is up-regulated in glioma initiating cells.

(A) Representative examples of dot plots and histograms of Rhod 123(–) subpopulations from 5 glioma cell lines were acquired by flow cytometry. Glioma cells (1 × 10⁷) were trypsinized and incubated with 0.1 μg/ml Rhod123 for 20 min in 37°C. After washing the cells were placed in 37°C for 90 min for Rhod123 exclusion in a dark compartment. Cells kept on ice to inhibit exclusion of Rhod123 were used as a positive control for gating in flow cytometry. Fractions of Rhod123(+) and Rhod123(–) cells were sorted using FACS Aria and a left panel shows a gating strategy. (B) Analysis of SPP1, OCT3/4 and NANOG gene expression in Rhod 123(–) subpopulations sorted from four human glioma cell lines. Total RNA was isolated from sorted cells using Qiagen RNeasy kit and the levels of SPP1, NANOG and OCT3/4 mRNA were determined by qPCR in Rhod 123(+) and Rhod 123(–) subpopulations; their expression in Rhod 123(+) subpopulations was taken as 1 (a red line). P values were calculated with a t-test and considered significant when *P ≤ 0.05 and **P ≤ 0.01. (C) Quantification of the expression of Spp1, Oct3/4 and Nanog in Rhod 123(–) subpopulations isolated from rat C6 glioma cells versus their levels in Rhod 123(+) subpopulations taken as 1 (a red line); n = 3.