Fig. 3.

Effects of LHN^TMZ, LHN^rutin, and LHN^TMZ+rutin on GSC phenotype of T98 G cells. The RNA expression of (A) CD133, (B) OCT4, (C) NANOG and (D) SOX2. (E) 3D-spheroid formation. T98 G cell spheres were formed in a 96-well, round-bottom, ultra-low attachment plate for 3 d Calcein-AM and PI staining identified the vitality of the spheres. Calcein-AM staining (green) indicates metabolically viable or proliferative regions, whereas PI staining (red) indicates the sites of necrosis or death. ImageJ software measured the size of captured tumor spheres and the ratio of the diameter of live/dead area. Scalebar: 150 µm. The RNA expression of (F) LOXL1-AS1, (G) HOTAIR, (H) H19 and (I) MALAT1. (J) Viability of T98 G cells after treatment with loaded LHNs by AO/PI staining. In morphological analysis, green cells with granular nuclei on one side showed apoptosis. In contrast, a circular nucleus evenly distributed in the center of the cell indicated that a cell is in interphase. Dark red cells with a vague rim indicated necrosis. Color-coded arrows indicate: live cells in white, early apoptosis in blue, late apoptosis in orange, and necrosis in red. (K) Analysis of apoptotic T98 G cells using Annexin V assay after loaded LHN's treatments. (L) The colony formation of T98 G cells after LHN^TMZ, LHN^rutin, and LHN^TMZ+rutin treatments. All values represent mean ± SD (n = 3). *P < 0.05, **P < 0.0001. CSC: Cancer Stem Cell, CD133: Prominin-1, OCT4: POU5F1, NANOG: Nanog Homeobox, SOX2: SRY-box 2, L: alive cells, A: apoptosis, N: necrosis.