Fig. 5: SMYD3 promotes NHEJ repair via interaction with NHEJ proteins and modulates LIG4/XRCC4/XLF dynamics at DNA damage sites.

A Schematic diagram of NHEJ fluorescent reporter repair in EJ5-U2OS cells. Two I-SceI sites were constructed between the whole GFP sites. Once transfected with I-SceI, cells will emit green fluorescence when repaired successfully through NHEJ pathway. B, C Flow cytometry analysis of GFP⁺ and DsRed⁺ cells of EJ5-U2OS cells transfected with I-SceI and indicated plasmids or siRNAs. The overexpression or knockdown efficiency of SMYD3 were determined by western blot as shown in the right graph. D Western blotting of the indicated proteins in WCLs from HEC1B cells stably expressing shRFP, shSMYD3-1, or shSMYD3-2. E, F Co-IP analysis of the indicated proteins in WCLs from HEK-293FT cells transfected with FLAG-SMYD3 treated with 10 Gy γ-ray irradiation (E) or VP16 at indicated concentration (F). G Dynamics of GFP-LIG4, GFP-XRCC4 or GFP-XLF accumulation at micro-irradiated sites in U2OS cells stably expressing shRFP, shSMYD3-1 or shSMYD3-2 (Left graph). Relative fluorescence intensity of GFP-LIG4, GFP-XRCC4 or GFP-XLF at micro-irradiated sites in the experiments described in the left graph (Right graph). Scale bar, 5 μm. H IP analysis of the mono/di-methylation or tri-methylation of the indicated proteins in WCLs from HEK-293FT cells transfected with GFP-LIG4, GFP-XRCC4, GFP-XLF or FLAG-SMYD3.