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. 2013 Oct 28;140(4):441–455. doi: 10.1111/imm.12154

Figure 1.

Figure 1

Silencing of thrombospondin 1 (TSP-1) and low-density lipoprotein receptor-related protein 1 (LRP1) differentially affects T-cell motility and adhesion. (a) The influence of transfection with scrambled control small interfering RNA (siRNA), LRP1 siRNA (alternative 1 see Materials and methods), TSP-1 siRNA (alternative 1 see Materials and methods) or both LRP1 siRNA and TSP-1 siRNA on the appearance of cells allowed to infiltrate a collagen gel for 30 min. (b) The influence of silencing, as described in (a), on cell shape expressed as per cent polarized cells. It is evident that silencing of LRP1 inhibits whereas silencing of TSP-1 enhances polarized cell shape. (c) Migration of cells into a collagen gel after transfection with scrambled control siRNA, LRP1 siRNA, TSP-1 siRNA or both LRP1 siRNA and TSP-1 siRNA. The number of cells at different depths was determined after 30 min. The results show that silencing of LRP1 as well as TSP-1 inhibits migration but that the silencing of LRP1 is more effective in this regard. (d) Adhesion of CD3-positive lymphocytes to intercellular adhesion molecule 1 (ICAM-1) and photographs showing pattern of spreading after transfection with control siRNA, TSP-1 siRNA and LRP1 siRNA as determined after 30 min. (e) Influence of an antibody to β2 integrin on adhesion of CD3-positive lymphocytes to ICAM-1 after transfection with LRP1 siRNA as determined after 30 min. (f) Influence of transfection with control siRNA and TSP-1 siRNA on anti-CD3-induced T-cell activation as determined by incorporation of radioactive thymidine after different times. (g, h) Influence of receptor-associated protein (50 μg/ml) on migration in collagen (g) and adhesion of CD3-positive cells to ICAM-1 (h). The results in the different figures represent mean values of three independent experiments or a representative experiment of three and four individual experiments (a, c, f). Between 86 and 98% of the cells in collagen in different experiments were identified as CD3-positive. **P < 0·01 versus control siRNA; ***P < 0·001 versus control siRNA.