Fig. 3. Treatment of DCs with calpain inhibitors results in accumulation of ß₂ integrin subunits, actin, vinculin and talin in podosomes. DCs plated overnight on poly-L-lysine-coated glass coverslips were left untreated or were treated with ALLM, ALLN, calpastatin peptide or the scrambled version of the calpastatin peptide (Calp Pept NC) and then fixed with 3% paraformaldehyde, permeabilised with 0.05% Triton X-100 and triple stained to detect ß₂ integrin subunit (green), actin (red) and vinculin (blue) (A-E). Inhibition of calpain with ALLM (B), ALLN (C) or the calpastatin peptide (E) resulted in an exaggerated accumulation of ß₂ integrin subunits, vinculin and actin in DC podosomes, compared with untreated cells (A) or cells treated with the scrambled version of the calpastatin peptide (D). ß₂ integrin subunit, actin and vinculin single staining from the boxed areas in main panels is shown magnified below. Quantification of the fluorescent intensity of the staining of ß₂ integrin (F), vinculin (G) and actin (H). Sizes of the actin core of podosomes of untreated and treated DC (I). Alternatively, DCs were stained to detect talin (green) and actin (red) (J-N). Inhibition of calpain with ALLM (K), ALLN (L) or the calpastatin peptide (N) induced an exaggerated accumulation of talin in DC podosomes, compared with untreated cells (J) or cells treated with the scrambled version of the calpastatin peptide (M). Single staining is shown to the right of panels J-N. (O) Quantification of the fluorescent intensity of the staining of talin. The micrographs are representative of the cytoskeletal organisation of DCs detected in three independent experiments. Bars, 10 µm. The graphs illustrate the mean percentage of cells ± s.e.m. from three experiments with 20 cells per treatment, per experiment. ^**P<0.01, ^***P<0.005 compared with values in the untreated cells (Student's t-test).