Fig 2: Effect of PAX6 on proliferation of GBM cells. (A) In U251 (left panel) and U118 (right panel) GBM cells, cell proliferation was measured with a microplate reader-based BrdU cell proliferation ELISA kit in cells transfected with the empty pcDNA3.1 plasmid and cells transfected with the PAX6 expression vector with or without TMZ treatment (400 µM) for 48 h. Cell proliferation was shown in OD values at 450 nm. aP<0.05 vs. pcDNA3.1; bP<0.05 vs. PAX6; cP<0.05 vs. TMZ + pcDNA3.1. (B) In U251 (left panel) and U118 (right panel) GBM cells, cell proliferation was measured with a microplate reader-based BrdU cell proliferation ELISA kit in cells transfected with Con-shRNA and cells transfected with PAX6-shRNA plasmids with or without TMZ treatment (400 µM) for 48 h. Cell proliferation was presented as the OD values at 450 nm. aP<0.05 vs. Con-shRNA; bP<0.05 vs. PAX6-shRNA; cP<0.05 vs. TMZ + Con-shRNA. PAX6, paired box 6; GBM, glioblastoma multiforme; TMZ, temozolomide; OD, optical density; Con, control; shRNA, short hairpin RNA.

Fig 3: Effect of TMZ/miR-223 signaling on the protein level of PAX6 in GBM cells. In (A) U251 and (B) U118 GBM cells with or without TMZ treatment (400 µmol/l) for 48 h, the protein levels of PAX6 were determined with western blot analysis in cells transfected with SCR (lanes 1 and 6), cells transfected with miR-223 mimic (lanes 2 and 7), cells transfected with miR-223 antagomir (lanes 3 and 8), cells co-transfected with miR-223 antagomir and PAX6-shRNA (lanes 4 and 9) and cells co-transfected with miR-223 mimic and PAX6 (lanes 5 and 10). ß-actin blotting was used as a loading control. Density of the PAX6 blot was normalized against that of the ß-actin blot to obtain a relative blot density, which was then expressed as fold changes to that of cells transfected with SCR without TMZ treatment (designated as 1). aP<0.05 vs. SCR; bP<0.05 vs. miR-223; cP<0.05 vs. miR-223 antagomir; dP<0.05 vs. miR-223 antagomir + PAX6-shRNA. TMZ, temozolomide; miR, miRNA; PAX6, paired box 6; GBM, glioblastoma multiforme; SCR, scrambled control; shRNA, short hairpin RNA.

Fig 4: Derivation and characterization of H9-NCCs differentiated from H9-hESCs. (A) Phase-contrast images of H9 hESCs at passage 56 (P56) and H9-NCCs and passage 0 (P0) and passage 4 (P4). (B) Flow cytometry analysis of H9-NCCs. (C) Immunocytochemistry analysis showed that H9 hESC-derived H9-NCCs were positive for HNK1, FOXD3, AP2a, p75, SOX9, SOX10 and were negative for neural progenitor marker PAX6 and undifferentiated hESCs markers SOX2 and OCT4. (D) Real-time PCR data showed the expression of OCT4, NANOG, SOX9, SOX10, P75, PAX3, ZIC1 and AP2a in H9-NCCs relative to H9 hESCs. Data were shown as the means ± SEM of three independent experiments. Unpaired two-tailed Student's t test. *p < 0.05, **p < 0.01. Scale bars, 100 µm.

Fig 5: Effect of TMZ/miR-223/PAX6 signaling on proliferation of GBM cells. In (A) U251 and (B) U118 GBM cells with TMZ treatment (400 µM) for 48 h, cell proliferation was measured at 48 h of culture with a microplate reader-based BrdU cell proliferation ELISA kit in cells transfected with SCR, cells transfected with the empty pcDNA3.1 plasmid, cells transfected with Con-shRNA, cells transfected with miR-223 mimic, cells transfected with miR-223 antagomir, cells transfected with PAX6, cells transfected with PAX6-shRNA, cells co-transfected with miR-223 mimic and PAX6 and cells co-transfected with miR-223 antagomir and PAX-shRNA. Cell proliferation was indicated by OD values at 450 nm. aP<0.05 vs. controls (SCR, pcDNA3.1 and Con-shRNA); bP<0.05 vs. miR-223; cP<0.05 vs. miR-223 antagomir; dP<0.05 vs. PAX6; eP<0.05 vs. PAX6-shRNA; fP<0.05 vs. miR-223 + PAX6. TMZ, temozolomide; miR, miRNA; PAX6, paired box 6; GBM, glioblastoma multiforme; SCR, scrambled control; Con, control; shRNA, short hairpin RNA; OD, optical density.