Pitfalls in AR42J-model of cerulein-induced acute pancreatitis
Fig 4
Transient transfection of AR42J cells.
A1-A3, transfection of AR42J with pEGFP-N1-Glo-I vector. Cells were seeded and incubated with 100 nM dexamethasone for 24 hours. Representative images at 20x magnification (upper line) and 5x magnification (lower line) indicate transfection efficacy by green colored cells in FITC-fluorescence channel (A2) and overlay (A3) compared to white light images (A1). B1-D2, transfection efficacy under different conditions, analyzed by Western Blot (WB) and qRT-PCR. Representative WB images are shown in B1. Elevated protein expression of Glyoxalase (Glo-I) was detectable after 6h and highest after 12 hours of transfection. Quantification (B2) of at least three independent experiments confirmed this increase to be significant. Controls, sham-treatment and the use of pEGFP-N1 vector confirmed the specific effect of the plasmid transfection. Higher amounts of plasmid concentrations deteriorated the expression of the target gene (D1-D2). Significant elevation of Glo-I expression was also confirmed on mRNA-levels by qRT-PCR (C). Glo-I: Glyoxalase-I. WB: Western Blot. Results are expressed as mean ± S.D. of at least three different experiments. Scale bars: 10μm (upper line), 100μm (lower line). * P<0.05, ** P<0.01, *** P<0.001.