Fig. 6: DNMT3A-silenced macrophages activate wild-type immune cells and cardiac cells by paracrine signaling.
From: Cell-intrinsic effects of clonal hematopoiesis in heart failure
a, Experimental procedure to analyze capacity of DNMT3A-silenced macrophages to activate wild-type macrophages. b, Expression of pro-inflammatory markers in human primary macrophages after indirect co-culture with DNMT3A-silenced macrophages (n = 12 biologically independent samples except for IL6 in siNC, n = 9, for IL12B in siNC and for CXCL10 in siDNMT3A, n = 10, four donors), P = 0.0193, 0.0339, 0.0008 and 0.0077 for IL1B, TNFA, IL12B and CXCL10, respectively. c, Visualization of procedure to analyze indirect activation of cardiomyocytes by DNMT3A-silenced macrophages via wild-type macrophages. d, Immunofluorescence quantification of cardiomyocyte cell size after treatment with supernatant from THP1-derived macrophages indirectly co-cultured with DNMT3A-silenced macrophages (n = 203 and 156 biologically independent cells for siNC and siDNMT3A, four independent experiments), P = 0.0007. e, Representative immunofluorescence analysis of cardiomyocytes stained with DAPI (blue) and phalloidin (green) to quantify hypertrophic effects (scale bar, 50 µm). f, Experimental procedure to analyze capacity of DNMT3A-silenced macrophages to activate wild-type T cells (top) and cardiac fibroblasts (bottom). g, Flow cytometry analysis of naive CD4+ T cells after indirect co-culture with DNMT3A-silenced human macrophages (n = 6 donors), P = 0.0370, 0.0480, 0.0280 and 0.0433 for IL4, IL17, GATA3 and T-bet, respectively. h, Representative immunofluorescence analysis of cardiac fibroblasts after treatment with supernatant from CD4+ T cells indirectly co-cultured with DNMT3A-silenced human macrophages. DAPI (blue), phalloidin (green), collagen type I (gray) and αSMA (red) are stained (scale bar, 50 µm). i, Immunofluorescence quantification of COL1A1 and aSMA in cardiac fibroblasts (n = 9 biologically independent samples, three independent experiments), P = 0.0242 and 0.0255 for αSMA and COL1A1. Data show means ± s.e.m. (b,d,i) or medians (g). Statistical significance was assessed by two-tailed unpaired (b,i) or paired (g) t-test and two-tailed Mann–Whitney test (d) (*P < 0.05, **P < 0.01, ***P < 0.001).
