Lentiviral transduction and transfection

Overexpression and short hairpin RNA (shRNA) -induced downregulation of IMPDH2 in CRC cells were achieved using the GV367 and GV248 shRNA lentiviral vector (genechem, Shanghai, China). The human shRNA sequences to inhibit IMPDH2 expression are listed as follows: IMPDH2 shRNA: GGACAGACCTGAAGAAGAA (genechem, Shanghai, China). The vectors were packaged in 293 cells. Recombinant lentiviruses were produced by transient transfection of HEK293T cells. Then, transduced cells were selected for 7 days with 0.6 mg/mL puromycin. Protein and mRNA of transfer cells were taken for qPCR and western blotting analyses.