Enhanced ICAM-1 and VCAM-1 adhesive function requires an appropriate tetraspanin environment. (A) Flow cytometry analysis of the expression of β₁ integrin (TS2/16 mAb), tetraspanins (anti-CD9 VJ1/20, anti-CD151 LIA1/1, anti-CD81 I.33.2.2, anti-CD82 TS82, and anti-CD63 TEA3/18 mAbs), ICAM-1 (HU5/3), and VCAM-1 (P8B1) in Colo320 cells (thin lines) or CD9 stably transfected Colo320 cell line (thick lines). Negative control PX63 is shown in dotted lines. (B) Heterotypic intercellular binding of K562 cell lines (parental and K562 α4 and K562 LFA-1 integrin transfectants) to Colo320 and Colo320CD9 cells transiently transfected with GFP-tagged versions of VCAM-1 and ICAM-1. Data are calculated as the ratio of double-positive aggregates (GFP-Colo320/CM-TMR K562) versus the nonaggregated GFP⁺ cells. Graph depicts the relative binding referred to the aggregation obtained with parental K562 cells (that ranged from 10% to 20% of the total GFP⁺ cells in the different transient transfections in the experiment shown) in a representative experiment out of 6 performed. (C) Heterotypic intercellular binding of K562α4 integrin–stable transfectant to VCAM-1–GFP transiently transfected Colo320 cells and the different chimeric clones of CD9/CD82. Data are calculated as the ratio of double–positive aggregates (GFP-Colo320/CM-TMR K562) versus the total number of GFP⁺ cells. Graph depicts relative binding referred to the aggregation of Colo320–VCAM-1–GFP cells in a representative experiment out of 3 (11% of the total GFP⁺ cells in the experiment depicted). On the right, flow cytometry analysis of the expression with anti-CD9 (10B1, which also recognizes the chimeric CD9/CD82 loop; thin lines) and CD82 (TS82; thick lines) of the different chimeric clones is shown.