Fig 2: Isolation of GSCs from U-87 MG and U251 cells. (A) The proportion of CD133+ cells in U-87 MG and U251 cells. (B) The sorted CD133+ U-87 MG cells were retested by flow cytometry after 7 days of culture, and the percentages of CD133+ cells and CD133- cells were shown. (C) CD133+ cells and CD133- cells were maintained in the serum-free condition for 7 days. CD133+ cells formed spheres, whereas CD133- cells showed degradation. Scale bar = 200 µm. (D) The sphere derived from sorted CD133+ cells was shown to express stem cell markers CD133 and Nestin. Scale bar = 20 µm. (E) CD133 and Nestin were overexpressed in proliferated sorted CD133+ cells compared with CD133- cells, whereas CD133- cells overexpressed LASS2 compared with CD133+ cells (*P < 0.05, vs. CD133- group; unpaired two-tailed Student's t-test; n = 3). (F, G) The glioma xenografts derived from CD133+ cells were more malignant than those derived from CD133- cells. (H) The average final tumor weight of the xenograft derived from CD133+ cells was significantly higher than that from CD133- cells in nude mice (F, G, and H, *P < 0.05, **P < 0.01, and ***P < 0.001, vs. CD133- group; unpaired two-tailed Student's t-test; n = 3 animals).

Fig 3: Analysis of LASS2 expression on a human glioma tissue microarry. (A) Representative immunohistochemical staining images of LASS2. Scale bar = 100 µm. (B) LASS2 levels in grade II to IV glioma samples were significantly lower than those in the normal adjancent tissue (NAT) (*P < 0.05 for grade II, and **P < 0.01 for both grade III and IV, n.s., no significance; one-way ANOVA). (C) The level of LASS2 in glioma of all grades was significantly lower than in NAT (*P < 0.05; unpaired two-tailed Student's t-test). (D) Representative images showing the immunuofluorescent co-staining of LASS2 and CD133 in the NAT and gliomas graded from I to IV. Scale bar = 20 µm.

Fig 4: The up-regulation of MCM2 enhanced the stemness of HCC cells.a Representative images of tumor-spheres in the MCM2 over-expression and control groups of the indicated cells, corresponding histogram and corresponding sphere morphology percentage chart. b Representative images of tumor-spheres formation in the limiting dilution assays of the indicated cells and the log-dose slope of the MCM2 over-expression and control groups. c Representative images of colony in the MCM2 over-expression and control groups of the indicated cells and corresponding histogram; d The immunoblotting for Bmi1, AKT1, CD133, ALDH1A1 and Epcam expression in the Vector and MCM2 groups of the indicated cells, determined by Western Blot assay; Data are shown as the mean ± SD from independent experiments. *P < 0.05.

Fig 5: TPTEP1 suppresses glioma cell stemness and radioresistance in vitro and in vivo. RT-qPCR analysis was performed to detect TPTEP1 expression levels in (A) SHG44 and U251 cells and in (B) stable/transient TPTEP1-depleted SHG44, stable TPTEP1-overexpressed U251 cells and the control cells. (C) Tumorspheres formation assay was performed in stable TPTEP1-depleted SHG44 cells, stable TPTEP1-overexpressed U251 cells and the control cells (×100 magnification). (D) Immunofluorescence analysis was performed to detect the expression and location of CD133 in transient TPTEP1-depleted SHG44 cells, transient TPTEP1-overexpressed U251 cells and their corresponding controls (×400 magnification). (E) Cell proliferation and radiosensitivity assays were performed in transient TPTEP1-depleted SHG44 cells, transient TPTEP1-overexpressed U251 cells and their corresponding controls. (F) Immunofluorescence analysis was performed to evaluate ?-H2AX foci formation in transient TPTEP1-silenced SHG44 cells, transient TPTEP1-overexpressed U251 cells and their corresponding controls. (G) Western blot analysis was performed to detect OCT4, ALDH1, ?-H2AX and H2AX expression levels in transient TPTEP1-silenced SHG44 cells, transient TPTEP1-overexpressed U251 cells and their corresponding controls. (H) A subcutaneous transplantation tumor model was applied to assess glioma cell proliferation regulated by TPTEP1 and R. *P<0.05, **P<0.01, ***P<0.001. TPTEP1, transmembrane phosphatase with tensin homology pseudogene 1; RT-qPCR, reverse transcription-quantitative PCR; OCT4, Octamer binding protein 4; ALDH1, aldehyde dehydrogenase 1; ?-H2AX, Histone H2AX; R, radiation.