Fig 2: Ccnd1 and Cdk4 expression in GBM cells cultured as neurospheres and as a monolayer (A) U87MG and T98G cells were grown as neurospheres (U87MG-N and T98G-N cells, respectively) or as monolayer cultures, and immunoblotting was carried out. β-actin was used as the loading control. Images show the results of one representative experiment out of three. Numbers above the blots represent the fold change of quantified value versus control normalized for β-actin. (B). Immunofluorescence images of neurospheres (left panel scale bar = 10 µm) or monolayer (right panel scale bar = 20 µm) stained for Ccdn1, Cdk4, Pxn, and Vcl. DAPI staining for nuclear detection. Original magnification, ×100. (C). Real-time RT-PCR assay for CD44, CD133, and NES. U87MG and T98G cells were transfected with vector or CCDN1 or CDK4R24C mutant expression vector. Data are expressed as means ± SD of three different experiments, each performed in triplicate. * p < 0.05 vs. vector. (D). T98G cells transfected with vector or CDK4R24C mutant expression vector were cultured as neurospheres for 72 h. Images show the results of one representative experiment out of three. Scale bar = 20 µm.