Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology
Article Title: Molecular association of CD98, CD29, and CD147 critically mediates monocytic U937 cell adhesion
Figure Lengend Snippet: Effect of pharmacological inhibitors of ERK, PKCδ, and actin polymerization on cell-cell aggregation or cell-fibronectin adhesion induced by ligation of surface adhesion molecules with aggregation-activating antibodies or immobilized fibronectin. (A left panel) U937 cells were incubated with pro-aggregative (activating) antibodies (1 µg/ml each as IgG1) to CD98 (AHN-18, 1 µg/ml), CD29 (MEM101A, 1 µg/ml), CD147 (M6-1D4, 10 µg/ml), and CD43 (161-46, 1 µg/ml) in the presence of chemical inhibitors to ERK (U0126, 25 µM), PKCδ (rottlerin, 10 µM), and actin polymerization (Cyto B: cytochalasin B, 10 µM) for 3 h. Aggregation of cells in the absence of stimuli (normal conditions) was less than 4%. Percentage of aggregation was quantitatively determined by cell-cell adhesion assays. (A right panel) Images of the aggregated cells in culture were obtained using an inverted phase-contrast microscope attached to a video camera, and captured using NIH image software. (B) U937 cells pretreated with 10 µg/ml of function blocking antibody to CD29 (P5D2) or inhibitors [U0126 (20 µM) and cytochalasin B (10 µM)] were seeded on fibronectin (50 µg/ml)-immobilized plates and further incubated for 3 h. Attached cells were determined by crystal violet assay, as described in Materials and Methods. (C) Cytotoxic activity of U0126, rottlerin, and CytoB was confirmed by a conventional MTT assay. Results (aggregation relative to control culture in the presence of stimuli or % of cytotoxicity) are expressed as mean±SEM from three independent experiments performed in triplicate. ** p
Article Snippet: Antibodies to CD98 (mouse, 4F2) and CD147 (rabbit, EMMPRIN) for immunoprecipitation and immunoblotting were from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and Sino Biological Inc. (Beijing, China), respectively.
Techniques: Ligation, Incubation, Microscopy, Software, Blocking Assay, Crystal Violet Assay, Activity Assay, MTT Assay