Fig. 5: NUSAP1 R422 promotes 5-FU resistance and cell proliferation, unlike R418.

A Western blot analysis confirming the expression of MYC-tagged wild-type (WT) NUSAP1, R418K, and R422K mutants in BGC-823-5-FU-R and SGC-7901-5-FU-R cells transfected with shNUSAP1 or control shRNA (shGFP). Tubulin was used as a loading control. B Cell viability assay over 7 days in NUSAP1-silenced cells expressing WT or mutant NUSAP1. ***p < 0.001. IC50 assays to evaluate the effect of WT or mutant NUSAP1 on 5-FU sensitivity in BGC-823-5-FU-R (C) and SGC-7901-5-FU-R (D) cells. Transwell migration assays showing the migratory ability of BGC-823-5-FU-R and SGC-7901-5-FU-R cells transfected with WT or mutant NUSAP1 after NUSAP1 down-regulation. Representative images are shown in (E), and quantitative analysis is presented in (F). Scale bar = 100 μm. ***p < 0.001. G, H Transwell invasion assays to assess invasive potential of cells transfected with WT or mutant NUSAP1 after NUSAP1 down-regulation. Representative images are shown in (G), and quantification is presented in (H). Scale bar = 100 μm. **p < 0.001. ***p < 0.001. I, J Subcutaneous xenograft assays in nude mice injected with cells expressing WT or mutant NUSAP1, with or without NUSAP1 knockdown. Representative tumor images and tumor weights will be shown. ***p < 0.001. Western blot analysis of R422me2 methylation levels in BGC-823-5-FU-R and SGC-7901-5-FU-R cells treated with increasing concentrations of PRMT1 inhibitors AMI-1 (K) and DCLX069 (L). Tubulin was used as a loading control. M Western blot analysis showing reduced R422me2 levels in BGC-823-5-FU-R and SGC-7901-5-FU-R cells transfected with shPRMT1 (shPRMT1-1# and shPRMT1-2#) compared to control. N Western blot analysis of R422me2 levels in cells expressing WT or methylation-deficient mutant (G80R) Flag-PRMT1 and MYC-NUSAP1, with or without PRMT1 knockdown, to confirm PRMT1 regulation of R422 methylation.