Effect of MAL-silencing on TCRζ and ZAP70 recruitment and phosphorylation at the immunological synapse. (A–C) Jurkat cells were transfected with either siCtr (A), or siMAL (B and C) in the absence (A and B) or the presence (C) of thapsigargin and allowed to spread for 3 min on an αCD3-coated coverslips. Cells were stained for pTCRζ and pZAP70 and analyzed by dSTORM-TIRF imaging. Top left insets depict the correspondent zx-stack widefield image projection of GFP-TCRζ. Right panels show a magnified image of a region of interest (frame). pTCRζ, green; pZAP70, red. (D–L) Population analysis in Jurkat cells processed as in A–C (n = 11) of the number of pTCRζ or pZAP70 clusters per square micrometer (D and E), the mean pTCRζ or pZAP cluster area per cell (F and G), the number of pTCRζ or pZAP70 clusters <100 nm for each cell analyzed (H and I), the number of pTCRζ or pZAP70 detections per individual cluster for each cell analyzed (J and K), and the percentage of clusters whose circularity is equal to one. Value is given by the ratio between the largest and the smallest feret diameters for all the clusters detected and plotted as the mean for each analyzed cell (L and M). Images representative of three experiments. ***, P ≤ 0.0001; **, P ≤ 0.01; *, P ≤ 0.05; Mann-Whitney test. Each circle represents a cell. Bar, 10 µm.