Immunofluorescence (IF) staining

In the study, CRC samples underwent fixation using 4% paraformaldehyde (Thermo Scientific, #FB002) and were subsequently embedded in Tissue-Tek O.C.T. compound (Sakura Finetek USA, Torrance, CA). Following this, 3 mm frozen sections underwent a blocking step with 3% donkey serum albumin in phosphate-buffered saline (PBS; Gibco, #10010031) for 30 min at 37 °C. For immunostaining, an anti-EFTUD2 rabbit polyclonal antibody (Proteintech, #10208-1-AP, 1:200) was diluted in PBS and incubated overnight at 4 °C. Subsequently, a goat anti-rabbit IgG Fluor Plus 647 secondary antibody (Thermo Scientific, 1:500, #A32733) was applied at room temperature for 1 h. Sections were then covered with Vectashield mounting medium containing 4’,6-diamidino-2-phenylindole (DAPI; Abcam, #ab104139). The obtained images were observed using a Confocal Laser Microscope System (Zeiss, LSM900), and the percentages were quantified using ImageJ software.

Simultaneously, cell samples (3000 cells per well) were fixed with 4% paraformaldehyde for 30 min and permeabilized with 0.1% Triton X-100 (Sigma, #T8787-100ML) for 15 min. After blocking with 3% donkey serum albumin, cells were incubated with primary mouse antibodies against EFTUD2 (Proteintech, #67855-1-Ig, 1:500) and primary rabbit antibodies against c-MYC (Proteintech, #10828-1-AP, 1:250) at 4 °C overnight. Subsequent staining involved goat anti-rabbit IgG Fluor 488 secondary antibody (Thermo Scientific, 1:500, #A11034) and goat anti-mouse IgG Fluor 568 secondary antibody (Thermo Scientific, 1:500, #A11004) at room temperature for 1 h. Nuclei were counterstained with DAPI, and images were acquired using a Confocal Laser Microscope System (Zeiss, LSM900).