FIG 4.

K848R mutation does not change CD133 degradation by the lysosomal pathway. U87MG cells were infected with a lentivirus containing CD133-WT or CD133-K848R. (A) Relative CD133 mRNA levels were determined by RT-PCR. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. (B) Results are presented as means and SD (n = 3). Statistical analysis was performed using the Student t test (ns, no significance). (C) Protein levels of CD133-WT and CD133-K848R were detected by Western blotting; β-actin was blotted as a loading control. (D) Cells were treated with 50 μM chloroquine or dimethyl sulfoxide (DMSO) for 12 h. The levels of CD133 were determined by Western blotting; β-actin was blotted as a loading control. (E) Cells were treated with 10 mM NH₄Cl or H₂O for 12 h. The levels of CD133 were determined by Western blotting; β-actin was blotted as a loading control. (F) An immunofluorescence assay was performed to examine the colocalization of CD133 (red) and LAMP1 (green). Bars = 20 μm. (G) Quantification of LAMP1-CD133 colocalization in confocal microscopy images by use of Pearson's coefficients. The fluorescence intensity signals in selected regions with similar areas were measured. Data are presented as means and SD for results from three independent experiments (n ≥ 20 cells) analyzed by the Student t test (ns, no significance).