Detection of CD133 (prominin‐1) in a human hepatoblastoma cell line (HuH‐6 clone 5)

Abstract

We examined CD133 distribution in a human hepatoblastoma cell line (HuH-6 clone 5). We directly observed the cultured cells on a pressure-resistant thin film (silicon nitride thin film) in a buffer solution by using the newly developed atmospheric scanning electron microscope (ASEM), which features an open sample dish with a silicon nitride thin film window at its base, through which the scanning electron microscope beam scans samples in solution, from below. The ASEM enabled observation of the ventral cell surface, which could not be observed using standard SEM. However, observation of the dorsal cell surface was difficult with the ASEM. Therefore, we developed a new method to observe the dorsal side of cells by using Aclar® plastic film. In this method, cells are cultured on Aclar plastic film and the dorsal side of cells is in contact with the thin silicon nitride film of the ASEM dish. A preliminary study using the ASEM showed that CD133 was mainly localized in membrane ruffles in the peripheral regions of the cell. Standard transmission electron microscopy and scanning electron microscopy revealed that CD133 was preferentially concentrated in a complex structure comprising filopodia and the leading edge of lamellipodia. We also observed co-localization of CD133 with F-actin. An antibody against CD133 decreased cell migration. Methyl-β-cyclodextrin treatment decreased cell adhesion as well as lamellipodium and filopodium formation. A decrease in the cholesterol level may perturb CD133 membrane localization. The results suggest that CD133 membrane localization plays a role in tumor cell adhesion and migration.

Subcellular Localization of CD133 and Interleukin-6 Receptor (IL-6R) in Human Hepatoblastoma Cell-Line (HuH-6 Clone-5)

“…CD133 is concentrated in microspikes, filopodia and the leading edge of lamellipodia in the fibroblasts expressing transfected CD133 [28]. Previously, we revealed that CD133 was preferentially concentrated in a complex structure comprising filopodia and lamellipodia [18]. In the present study, IL-6R was also concentrated at the similar site.…”

“…According to a method previously reported by Akita et al [18], cells were cultured to near confluence at 37°C in 35 mm culture dishes containing DMEM supplemented with 10% FBS. The cultures were treated with 2 concentrations of CD133 antibody (1 and 10 μg/mL treatment groups, 4 dishes per concentration) in fresh culture medium at 37°C in 5% CO 2 for 48 h. One group is the control group and receives no treatment.…”

Development of In Vitro Method for Assaying Anti-Angiogenic Effect of Drugs

“…Our study also demonstrated that lovastatin abrogated the cell-cell adhesion and capillary tube degradation [11]. Cholesterol chelating agent, methyl--cyclodextrin diminished cell adhesion by decreasing desmosomes and intercellular digitations [162]. According to the recent study of Koyama-Nasu et al, [161], CD133 interacts with plakoglobin (desmosomal linker protein).…”

Detection of the Hematopoietic Stem and Progenitor Cell Marker CD133 during Angiogenesis in Three-Dimensional Collagen Gel Culture

“…In addition, we reported that the cholesterol chelating agent, methyl- β -cyclodextrin, diminished cell adhesion by decreasing desmosomes and intercellular digitations. A decrease in the cholesterol level may perturb CD133 membrane localization [50]. Modulation of cell-cell adhesion may help to explain the degradation of the capillary tubes.…”