Overexpression of FBXL14 promoted GSC differentiation and inhibited GSC tumor growth. (A) IB analysis of FBXL14, c-Myc, SOX2, GFAP, and MAP2 in the GSCs transduced with Flag-FBXL14 expression through lentiviral infection during a time course of differentiation. Forced expression of FBXL14 in GSCs accelerated expression of differentiated cell markers (GFAP and MAP2) and augmented loss of GSC markers (c-Myc and SOX2) during serum-induced GSC differentiation. VEC, vector. (B–E) Immunofluorescent staining of Flag-FBXL14 and c-Myc (B) or GFAP (C) in GSCs (T387) expressing Flag-FBXL14 or vector control during a time course of GSC differentiation induced by serum (FBS). Cells were counterstained with DAPI (blue) to mark nuclei. Quantifications show that forced expression of FBXL14 significantly accelerated the reduction of c-Myc–positive cells (D) and increased percentage of GFAP-positive cells (E) during serum-induced GSC differentiation. n = 4. Data are from three independent experiments, and representative images are shown. (F–I) The effect of FBXL14 overexpression on GSC tumorsphere formation. GSCs (T387) were transduced with Flag-FBXL14 or vector control. (G) Ectopic expression of Flag-FBXL14 reduced tumorsphere formation of GSCs. (H and I) Quantifications show that forced expression of FBXL14 significantly decreased GSC tumorsphere size (H) and number (I). n = 3–4. Data are from three independent experiments, and representative images are shown. (J) Growth curves of GSCs expressing Flag-FBXL14 or vector control. GSCs (T387) were transduced with Flag-FBXL14 or vector control through lentiviral infection and then measured for cell growth over a time course. Ectopic expression of FBXL14 significantly inhibited GSC growth. n = 4. Data are from three independent experiments. Data are mean ± SD. ***, P < 0.001. Student’s t test was used to assess the significance. (A and F) Mass is shown in kilodaltons.