Fig. 1

Morphological characteristics of Nck1 knocked-out and Jurkat T cells. a CRISPR/Cas9-mediated Nck1 deletion in Jurkat T- cells. Jurkat T cells were transfected with a plasmid containing a guide RNA against nck1. A stable clone of Nck1-knockout (N1KO) cells was generated using the limiting dilution technique. Plasmids containing human Flag-tagged wild-type Nck1 were transfected into Nck1-knockout cells. The expression of Nck1 was verified by western blot using antibodies against Nck1, Nck2, and GAPDH (left panel). The band intensity of each protein was quantified using ImageJ software and is presented as the ratio of Nck1 to GAPDH. Data are presented as mean ± SD from five independent experiments, n = 5 (right panel). b Nck1-knockout cells (N1KO) had CD3 expression level comparable to that of control Jurkat T-cells. N1KO and Jurkat T cells were stained for surface CD3 molecules with PerCP-conjugated anti-human-CD3ε antibody followed by analysis with flow cytometry. Data are presented as histograms (left panel) and bar graphs of mean fluorescence intensity (MFI; right panel) from three independent studies (n = 3). c Deletion of Nck1 causes morphological changes. Cells were left untreated or treated with anti-CD3 antibody (OKT3) alone for 5 min or pre-treated with AX-024 for 30 min, followed by OKT3 stimulation for 5 min. Unlabelled cells were observed under a holotomographic microscope. One representative image from three independent studies of 3D holotomographic (left) and maximum intensity projection (MIP) (right) images is shown, n = 3 (top panel). Cell circularity was quantified using ImageJ software and presented as the circularity index (A.U) from three separate experiments, n = 3 (button panel). ns = non-significant; **p < 0.01; ***p < 0.001; ****p < 0.0001