Fig. 2. Leucine-rich repeat kinase 2 (LRRK2) regulates the stemness and tumorigenesis of glioma stem cells (GSCs). (A) Representative western blot images of phospho-LRRK2 (pLRRK2), LRRK2, stemness factors (CD133, NESTIN, OLIG2, SOX2), and differentiation marker glial fibrillary acidic protein (GFAP) in control 0317 cells (1, 0317-shControl), and two different LRRK2-knockdown 0317 cell lines (2, 0317-shLRRK2-1; 3, 0317-shLRRK2-2). (B) Limiting dilution assays (LDAs) performed using 0317-shControl, 0317-shLRRK2-1, and 0317-shLRRK2-2 cells (**p<0.01, t-test). (C) Cell proliferation assays performed using 0317-shControl, 0317-shLRRK2-1, and 0317-shLRRK2-2 cells. All error bars represent mean±SEM (n=3). **p<0.01, t-test. (D) Representative western blot images of pLRRK2, LRRK2, stemness factors (CD133, NESTIN, OLIG2, SOX2), and differentiation marker GFAP in control 448T cells (1, 448T-shControl), and two different LRRK2-knockdown 448T cell lines (2, 448T-shLRRK2-1; 3, 448T-shLRRK2-2). (E) LDAs performed using 448T-shControl, 448T-shLRRK2-1, and 448T-shLRRK2-2 cells (**p<0.01, t-test). (F) Cell proliferation assays performed using 448T-shControl, 448T-shLRRK2-1, and 448T-shLRRK2-2 cells. All error bars represent mean±SEM (n=3). **p<0.01, t-test. (G) Magnetic resonance imaging images of the whole brains from mice implanted with 448T-shControl, 448T-shLRRK2-1, or 448T-shLRRK2-2 cells. (H) H&E staining of the whole brains from mice implanted with 448T-shControl, 448T-shLRRK2-1, or 448T-shLRRK2-2 cells. Scale bars=100 μm. (I) Kaplan–Meier survival plots for the orthotopic xenograft mouse model (log-rank test). The shLRRK2 treated orthotopic mouse models showed dramatically decrease of tumor size and increase of overall survivals.