Fig. 1
From: Tailoring a novel colorectal cancer stem cell-targeted therapy by inhibiting the SMYD3/c-MYC axis
Analysis of SMYD3 involvement in cancer stemness and other cancer features in CRC models. a In vitro binding assay between HIS-SMYD3 and untagged recombinant c-MYC protein. GST-HSP90 C-terminal (616–736) was used as a positive control. b Upper panel: scheme showing the four FLAG c-MYC (WT + M1-M3) fusion proteins used in this study. Lower panel: in vitro binding assay between HIS-SMYD3 and recombinant FLAG c-MYC fragments. c Upper panel: Scheme showing the position of the purified P14, P14ext1, and P14ext2 peptides, which are located in the c-MYC transactivation ___domain (TAD). Lower panel: in vitro competition assay. HIS-SMYD3 bound to histidine beads was incubated with untagged c-MYC recombinant protein in the presence of escalating doses (0, 1, 5, 25, 125 µM) of the purified P14, P14ext1, and P14ext2 peptides. Bound proteins were visualized by immunoblot using anti-c-MYC and anti-HIS antibodies. d Co-immunoprecipitation assay of endogenous SMYD3 and c-MYC in HCT-116 CRC cells. e−h Tumorsphere formation assay of WT and SMYD3-KO HCT-116 cells: tumorsphere brightfield imaging (e, scale bar: 500 μm), number (f, day 8), area (g), and diameter (h) at day 3 to 8. i Area (left panel) and brightfield imaging (right panel, scale bar: 500 μm) of replated WT and SMYD3-KO HCT-116 tumorspheres. j Results of the Gene Ontology (GO) enrichment analysis of differentially expressed (DE) genes in SMYD3-KO vs WT HCT-116 cells. *p < 0.05 SMYD3-KO vs WT parental cells. HIS-PD= His-Pull down. Where applicable, data are expressed as means ± SD of 3 independent experiments
