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. 2016 Dec 22;8(10):16340–16355. doi: 10.18632/oncotarget.14092

Figure 6. Stable knockdown of Spp1 expression in C6 glioma cells reduces sphere formation and stemness factor expression.

Figure 6

(A–B) Efficient stable knockdown of Spp1 expression in rat C6 glioma cells was confirmed by qPCR and ELISA. Control shNeg cells expressed Spp1 and SPP1/osteopontin at the similar level as parental C6 cells (WT). (C) Stable knockdown of Spp1 expression in C6 glioma cells inhibited formation of spheres. Cells were seeded at a low density (4000 cells/ml) onto non-adherent plates and cultured in DMEM/F-12 medium, supplemented with B27, 20 ng/ml bFGF, 20 ng/ml EGF and antibiotics. Representative images show the reduced formation of spheres (100–200 μm in size) derived from the shSpp1 glioma cells 14 days after seeding. (D) Quantification of resulting spheres derived from shNeg and shSpp1 glioma cells was performed 14 days after seeding; data are means ± s.d., n = 3. (E) Reduced expression of Nanog and Oct3/4 in spheres derived from shSpp1 glioma cells. Spheres were collected by centrifugation 14 days after seeding and the expression of Nanog and Oct3/4 in cultures was determined by qPCR; data are presented as means ± s.d., n = 3.