Fig. 1

SMARCA4 is a major H4R3me2a-associated protein and promotes CRC cell proliferation. a Immunoaffinity purification to identify proteins that are associated with H4R3me2a. The protein bands were retrieved and analyzed by mass spectrometry. SMARCA4 peptide fragments identified by mass spectrometric assay (right). b Peptide pull-down assay to detect the interactions between H4, H4R3me2a, and H4R3me2s peptides and SMARCA4 in HCT116 cell nuclear extracts (top panel). Coomassie staining shows equivalent loading of the three peptides (middle panel). The modification of the synthesized peptide was confirmed by dot blot analysis with specific antibodies (bottom panels). c Peptide pull-down experiments were performed with H4, H4R3me2a, and H4R3me2s peptides and purified recombinant glutathione S-transferase (GST) fusion proteins of SMARCA4 fragments expressed in E. coli. as in b. d MST assay to identify direct interactions between SMARCA4-F4 and H4R3me2a peptides. The dissociation constant (Kd) between SMARCA4-F4 and H4R3me2a peptide is 5.36 ± 0.26 μM. e, f Identification of the effect of SMARCA4 knockdown (SMARCA4-KD) in HCT116 cells by quantitative real-time PCR (e) and western blot analysis with indicated antibodies (f). Hsp70 served as a loading control. g Proliferation of HCT116 cells following knockdown of SMARCA4. Values at the indicated time points represent mean ± s.d. from three independent tests; **P < 0.01. h EdU proliferation analysis of the effect of siRNA knockdown of SMARCA4 on the growth of HCT116 cells. Representative images (left panel) and quantitative analyses of the assay (right panel) are shown. i Colony formation assay of HCT116 cells following SMARCA4 knockdown. Representative images (left panel) and quantitative analyses of the colony formation (right panel) are shown. j Migration assay of HCT116 cells following SMARCA4 knockdown. The numbers of migrated cells were quantified by counting the numbers of cells in entire fields at ×200 magnification. Representative images (left panel) and quantitative analyses of the migrated cells (right panel) are shown. For e, h, i, and j, results are shown as mean ± s.d. from three independent experiments; **P < 0.01 compared with the scrambled negative control (NC)