Tetracycline-inducible protein expression in pancreatic cancer cells: Effects of CapG overexpression

Figure 2 Identification of stable pN1βactin-rtTA2S-M2-IRES-EGFP- expressing Panc-1 (A) and MiaPaca (B) cell clones and their functionality (C). Panc-1 and MiaPaca cells were transfected with pN1βactin-rtTA2S-M2-IRES-EGFP and stable expressors selected using 300 μg/mL G418. Clones were isolated and transfected with pTRE2hygluc, used as an indirect measure of rtTA activity. Cells were treated for 24 h with 500 ng/mL doxycycline (black bars) or an equivalent volume of PBS (white bars) and luciferase activity measured in 50 μg protein (1 mg/mL protein; 50 μL was assayed per sample). A: 90 G418 resistant clones were isolated from Panc-1 cells, of which 25% showed > 5-fold increase in luciferase activity in the stimulated (black bars in Figure 1) compared to the unstimulated (white bars in Figure 1) condition; B: Of 170 G418-resistant cells identified in MiaPaCa-2 cells, 60% showed > 5-fold increase in luciferase expression; C: Two stable pN1βactin-rtTA2S-M2-IRES-EGFP Panc-1 (Pβtet41 and Pβtet86) and MiaPaCa-2 (Mβtet110and Mβtet132) cell lines were transiently transfected with the pTRE2hygCapG vector and treated for 24 h with or without 500 ng/mL doxycycline. Cell lysates was subjected to Western Blotting for the detection of CapG. β-actin and GAPDH were used as loading controls, respectively. Tet: Tetracycline.