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. 2019 Aug 8;10(8):596. doi: 10.1038/s41419-019-1836-2

Fig. 4. Folic acid promotes U251 tumor spheres stemness.

Fig. 4

a TS overexpress SOX2 and CD133 as shown by immunohistochemistry, n = 3. Scale bar = 20 μm. b TS differentiate into neurons, oligodendrocytes and astrocytes as indicated by immunofluorescence staining with GFAP, NeuN and OLIG2, respectively, n = 3. Scale bar = 10 μm. c TS overexpress NANOG, SOX2, and OCT4 transcripts as shown by RT-qPCR. Addition of folic acid (0,5 mM) further increases the expressions of NANOG, SOX2, and OCT4 in TS by comparison with ML; RNA was extracted from 3 independent cell cultures then gene expression was measured by 3 independent RT-qPCR and normalized for the geometric mean of TBP, RPL13a, and HMBS. d Folic acid rescues TS formation when added at a concentration ≧ 0.1 mM in methionine-free media. Cells are grown in methionine free DMEM which already contains 0.009 mM folic acid then folic acid is added to reach the indicated final concentrations. The MET sample is used as a reference and represents cells grown in DMEM containing 0.01 mM methionine. Samples are compared to cells grown in 0.009 mM folic acid, n = 9; *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant; ML; monolayer cells, TS: tumor spheres;