Fig 2: 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 3: LASS2 inhibits sphere formation of GSCs. CD133+ U-87 MG cells were stably transfected with pLV and pLV-LASS2. (A) Downregulation of LASS2 promoted sphere formation of GSCs (**P < 0.01, vs. pLV control; unpaired two-tailed Student's t-test; n = 3). Scale bar = 200 µm. (B) Downregulation of LASS2 significantly reduced the protein level of LAAS2 while significantly increasing those of Notch1 and glioma stem cells stemness proteins CD133, Nestin and Sox2 (*P < 0.05, vs. pLV control; unpaired two-tailed Student's t-test; n = 3).

Fig 4: 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 5: Effect of LASS2 on cell migration, invasion and apoptosis. (A) Wound healing assay of pLV-vector or pLV-LASS2-transfected glioma/glioblastoma cells at 0 h, 12 h and 24 h after scratch. The images were taken from an inverted microscope under 10× magnification (*P < 0.05 and **P < 0.01, vs. pLV control; unpaired two-tailed Student's t-test; n = 3). Scale bar = 200 µm. (B) Colony formation assay in pLV-vector or pLV-LASS2-transfected U251 and U-87 MG cells. Images were acquired at 4× magnification (*P <0.05, vs. pLV control; unpaired two-tailed Student's t-test; n = 3). (C) Transwell assay demonstrated that LASS2 inhibits the migration of U251 and U-87 MG cells compared with the pLV control (*P <0.05; unpaired two-tailed Student's t-test; n = 3). Scale bar = 200 µm. (D) The immunofluorescence staining of MMP9 and SPHK1 was shown in both U251 and U-87 MG cells. Scale bar = 200 µm. (E) RNA-Seq shows that LASS2 influenced cell migration/invasion, apoptosis, epithelial- mesenchymal transition (EMT) conversion and cellular life activity. (F) Overexpression of LASS2 reduced the protein levels of MMP2, MMP9, and SPHK1 while increasing that of TIMP2 in both U251 and U-87 MG cells. (G) LASS2 overexpression increased the levels of Bax, cleaved Caspase-3, TNF-a, and p53 while reducing that of Bcl-2 in both U251 and U-87 MG cells. (H) Overexpression of LASS2 reduced the protein levels of Vimentin and N-cadherin while increasing that of E-cadherin in both U251 and U-87 MG cells (F, G, and H, *P <0.05 and **P < 0.01, vs. pLV control in each cell line; unpaired two-tailed Student's t-test; n = 3).