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. 2014 Aug 6;5:4619. doi: 10.1038/ncomms5619

Figure 4. H3K9 methylation levels depend on methyltransferases and demethylases and the tensed cytoskeleton.

Figure 4

(a) Representative images of western blotting of G9a or SUV39h1 expression after siRNA knockdown. Control melanoma cells were transfected with two sets of G9a/SUV39h1 siRNAs (1 and 2), respectively, and western blotting assays were performed to confirm the knockdown efficiencies to be 48% (1 siRNA) and 45% (2 siRNA) for G9a, and 58% (1 siRNA) and 43% (2 siRNA), respectively. Three independent experiments showed similar results. (b) Silencing or inhibiting demethylase KDM7 increases H3K9 methylation of control melanoma cells in soft fibrin gels. Control cells were transfected with H3K9 FRET biosensors and KDM7 siRNAs (1 and 2) or negative control siRNA, or treated with KDM7 inhibitor daminozide. The cells were seeded into 3D soft fibrin gels and H3K9 methylation levels were measured after 3, 19 or 48 h. Mean±s.e.m.; n>40 cells for each data point; *P<0.05. (c) Knockdown of G9a or SUV39h1 decreases the H3K9 methylation in control B16-F1 cells. (d) Inhibiting microtubule, myosin light chain kinase (MLCK) or actin polymerization decreases H3K9 methylation. Control B16-F1 cells were incubated with Colchicine (Colch, 10 μM for 30 min) to inhibit microtubule polymerization, ML7 (25 μM for 30 min) to inhibit myosin light chain kinase, or Latrunculin A (LatA, 1 μM for 30 min) to disrupt actin filaments. Mean±s.e.m; n>40 cells for each data point; *P<0.05; **P<0.001.