Fig. 5

Nck1 depletion and Nck–CD3 interaction inhibition affected on actin polymerization in Jurkat T cells. a Deletion of Nck1 and inhibition of Nck–CD3 association impaired actin polymerization. Cells were untreated or stimulated with 5 μg/mL of anti-CD3ε antibody (OKT3) in the presence or absence of 10 nM AX-024. Cells were stained for fibrous-actin filament with rhodamine-phalloidin-conjugated FITC (green) then observed under holotomographic microscope (Tomocube). One representative picture of each sample was cropped for presentation purpose and three independent experiments were done, n = 3, (left panel). ImageJ software was used to measure the value of integrated density, area of selected cell, and mean fluorescence of background before calculating the Corrected total fluorescence (CTCF) intensity. Data represented the mean ±  SD of CTCF intensity from three separated experiments, n = 3 (right panel). b Decreased F-actin/G-actin ratio was found in Nck1-depleted cells and Nck–CD3 interaction inhibition. Cells were treated as above and fractions of G-actin and F-actin were prepared before subjecting to immunoblot analysis using antibody against actin. One representative immunoblot of three independent studies was shown (left panel). Band intensity of G-actin and F-actin were quantified using ImageJ software, and the ratio of F-actin/G-action was then calculated. Data are represented as the mean ± SD of band intensity from three independent experiments, n = 3 (right panel). c Re-expression of wild-type Nck1 in Nck1 knocked-out (N1KO) cells rescued actin polymerization. Cells were treated and stained with rhodamine-phalloidin-conjugated FITC (green) as indicated above followed by observing under the fluorescent microscope. One representative fluorescent image of three independent experiments were shown, n = 3, (left panel). ImageJ software was used to analyse for the CTCF intensity. Data are represented as the mean ± SD of CTCF intensity from three separated experiments, n = 3 (right panel). ns = non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001