Fig. 4: Decitabine stabilizes the immune synaptic cleft by strengthening the actin cytoskeleton. | Nature Communications

Fig. 4: Decitabine stabilizes the immune synaptic cleft by strengthening the actin cytoskeleton.

From: Epigenetic modulation of immune synaptic-cytoskeletal networks potentiates γδ T cell-mediated cytotoxicity in lung cancer

Fig. 4

a Immunofluorescence staining of F-actin (red), ICAM-1 (green), pTyr (phosphotyrosine, white) at immune synapses between γδ T cells and DAC-pretreated H1299 lung cancer cells at D3R3. DAPI: 4′,6-diamidino-2-phenylindole. Scale bar: 10 μm. Three independent experiments were performed. b Immunofluorescence images of the interfaces between γδ T cells and H1299 lung cancer cells (parental vs. ICAM1 knockout (KO-ICAM1)). Signals of F-actin (red) in the periphery of H1299 cancer cells are shown in two-and-a-half-dimensional (2.5D) images in the lower panels. Scale bar: 10 μm. Three independent experiments were performed. c Dot plots of signal intensities of F-actin and ICAM-1 from five pTry-positive immune synapses between γδ T cells and H1299 lung cancer cells (parental or KO-ICAM1) from three independent experiments (mean ± SD). d Immunofluorescence images of immune synapses between γδ T cells (marked with T) and H1299 lung cancer cells (marked with C) stained for ICAM-1, F-actin, and pTyr. Three independent experiments were performed. e Dot plots of F-actin signal intensities at immune synapses between γδ T cells and H1299 cells. H1299 cells are pretreated with PBS (Mock), DAC alone or a combination of DAC pretreatment (D3R3) and 1 μg/mL Cyto B (cytochalasin B) for 1.5 h before coculture with γδ T cells (mean ± SD). n = 13–20 immune synapses over three independent experiments. f Immunofluorescence images of immune synapses between γδ T and H1299 cells pretreated with PBS (Mock), DAC alone, and combination of DAC and Cyto B. Blow-up images of the square areas for each treatment are shown in the lower panels. Arrows denote immune synapses between γδ T and H1299 cells. Scale bar: 100 μm (upper) and 20 μm (lower panels). Two independent experiments were performed. g Dot plots showing numbers of immune synapses per cancer cell on eight randomly taken high power fields for H1299 cells pretreated with PBS (Mock), DAC, and combination of DAC and Cyto B (mean ± SD). The p value is calculated by the two-way ANOVA (c) or one-way ANOVA with Tukey’s multiple comparisons test (e, g).

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