Rescue experiment excluded the possible off-target for GCK shRNA. (A) C-terminal Myc-tagged GCK WT or GCK shRNA-resistant allele M5 expression construct was generated based on PCDH-MCS-EF1-eGFP lentiviral vector as stated in the Methods section. (B) Tet-On-shGCK MM.1S cells were infected with PCDH-EV, PCDH-GCK-myc, or PCDH-GCK(M5)-myc lentiviral particles. Cells were selected by GFP and treated with Dox 400 ng/mL for 2 days to turn on shRNA. Cell lysates were analyzed by western blot for GCK, IKZF1, and c-MYC expression. Selected cells from panel B were cultured with Dox 400 ng/mL for 5 days and cell proliferation was measured by (C) MTS assay, (D) Annexin V and 7-AAD staining for apoptosis analysis, and (E) PI staining for cell-cycle analysis.