1. Nuclear Arp3 mediates formation of TCR-induced nuclear actin filaments.
Nuclear Arp3 mediates formation of TCR-induced nuclear actin filaments. (A and B) Silencing of Arp3 expression impairs formation of TCR-induced nuclear actin filaments. (A) Representative images of Jurkat NLA cells expressing the indicated shRNAs after PMA/Iono stimulation. (B) Relative occurrence of cells with nuclear F-actin (top) and Western blot expression analysis (bottom). GAPDH, glyceraldehyde phosphate dehydrogenase. (C and D) Pharmacological inhibition of Arp2/3 complex impairs formation of TCR-induced nuclear actin filaments. Jurkat NLA cells were treated with DMSO or increasing amounts of the Arp2/3 inhibitor CK-869, stimulated with PMA/Iono, and analyzed for formation of nuclear actin filaments. (C) Representative images. (D) Relative occurrence of cells with nuclear F-actin. (E to G) Inhibition of nuclear Arp2/3 impairs formation of TCR-induced nuclear actin filaments at the IS. Primary human CD4+ T cells expressing mCherry or nucleus-targeted dn Arp2.mCherry (nuc.dnArp2.mCherry) together with nuc.lifeact.GFP were incubated with SEB-loaded Raji B cells (red) analyzed for the formation of F-actin in the nucleus and at the cell-cell contact. (E) Representative micrographs. Scale bar, 5 μm. Yellow arrowheads indicate F-actin at the IS, and white arrowheads indicate F-actin in the nucleus (see also movie S9). (F) Quantification of cells with nuclear F-actin. (G) Quantification of cells with F-actin at the IS. D1, donor 1. Silencing of NIK (H and I) or NWASP (J and K) expression impairs formation of TCR-induced nuclear actin filaments. (H and J) Representative images of Jurkat NLA cells expressing the indicated shRNAs after PMA/Iono stimulation. Quantifications (B, D, I, and K) depict means ± SD from three experiments with at least 30 cells evaluated each per condition. Statistical significance relative to shC (B, I, and K) and DMSO-treated (D) controls was assessed by one-sample t test.
N. Tsopoulidis et al. Sci. Immunol. 2019;4:eaav1987
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