|Supplementary figure legends
Supplementary Figure 1: (A) Expression of the stemness markers Oct4 and Nanog was assessed by immunofluorescence in self-renewing and serum-differentiated TG1 cells. Nuclei were stained with DAPI. (B) Expression of the miR-302-367 cluster was determined by QPCR analysis after 4 days of serum-mediated differentiation using Taqman Low Density Array (TLDA). ND refers to “not detected”, CT to cycle threshold. Error bars are derived from 3 independent experiments. (C) QPCR analysis of miR-302-367 cluster expression during serum-mediated differentiation. Self-renewing TG6 cells were cultured either in their defined medium or in 0.5% serum to induce differentiation. Total RNA was extracted at different time points (1, 2, 3, 8, and 15 days), and the expression of miR-302a, b, c, d, and 367 was determined using Taqman probe as described in the Materials and Methods section. (D) Expression of miR-302-367 cluster members in two other self-renewing primary cell lines obtained from glioblastoma (GB1 and #1056).
Supplementary Figure 2: (A) TG1 cells were transfected either with the non-relevant control siLUC or with anti-miR-302a, anti-miR-302b, or anti-miR-367. The day following the transfection, the cells were treated with serum to induce differentiation. (B) An identical experimental setting was used to transfect self-renewing TG1 cells either with the control siLUC or with a pool of the three anti-miR (anti-miR-302a, anti-miR-302b, and anti-miR-367). After 3 days, miR-302a, miR-302b, and miR-367 expression levels were assessed by QPCR using taqman probes and compared to their relative expression in self-renewing TG1. Error bars are derived from 3 independent experiments.
Supplementary Figure 3: (A) Expression of stemness markers (SHH, Oct4, Nanog) was assessed by immunofluorescence in TG6 Ctrl (upper panels) and TG6 Cluster 302 (lower panels) cells cultured in growing medium containing EGF and bFGF (NS34+) – required for GiCs proliferation and self-renewal. Expression of glioneuronal markers (GFAP and NeuN) was assessed by immunofluorescence in TG6 Cluster 302#1 cells cultured in NS34+ (B) or after 4 days of culture in medium depleted of EGF and bFGF (NS34-) (C). Nuclei were stained with DAPI. (D) TG6 Ctrl and TG6 Cluster 302#1 cells were seeded at a density of 10 cells/well in a 96 wells plate and allowed to grow. After 1 month the neurospheres were counted; 10 neurospheres/well corresponded to a clonal efficiency of 100%. Error bars are derived from 3 independent experiments. Statistical analysis was performed using Student’s t-test; **** corresponds to a P-value of 10-14 and indicates a highly signiﬁcant difference between the clonal efficiency of TG6 Ctrl and TG6 Cluster 302#1 cells. (E) TG6 Ctrl and TG6 Cluster 302#1 cells stably expressing the red fluorescent protein, were seeded on the top surface of organotypic brain slice culture of mouse brain, as described in the Materials and Methods section. After 1 month, each organotypic culture was embedded in paraffin and sliced. Cell infiltration and growth within the neural host tissue was visualized by tracking the red fluorescent protein. Dashed lines mark the neural tissue boundaries. The histogram represents a measurement of the fluorescence intensity quantified using the Image J software. The values are compared to the background fluorescence measured in the same conditions in unseeded mouse brain slices.
Supplementary Figure 4: (A) TG6 Cluster 302#1 cells were transfected either with a non-relevant siRNA siLUC or with a pool of anti-miR specific for miR-302a and 302b (clear miR-302a/b). Total RNA was extracted and CXCR4 mRNA levels were assessed by QPCR. The results are expressed as relative expression compared with siLUC transfected TG1 Ctrl cells. Error bars are derived from 3 independent experiments. (B) CXCR4 protein expression was assessed by immunofluorescence in TG6 Ctrl and TG6 Cluster 302#1 cells. Nuclei were stained with DAPI. (C) Boyden chambers assays were performed using TG6 Ctrl or TG6 Cluster 302#1 cells in the presence or absence of the CXCR4 ligand SDF1. (D) Boyden chambers assays were performed using TG6 Ctrl cells, TG6 Cluster 302#1 cells, and TG6 Cluster 302#1 cells in which CXCR4 expression was restored by a CXCR4 construct lacking the 3’UTR sequences.