Transient Transfection of AKR-2B Mouse Fibroblasts

AKR-2B mouse embryo fibroblasts (MEFs) were seeded at 4.0 × 10⁴ in McCoy’s 5A medium supplemented with 5% heat-inactivated FBS in 6-well plates. Cells were incubated at 37°C in an incubator with 5% CO₂ for 24 h to facilitate adherence to the plate. MEFs were transiently transfected with a total of 2 μg/mL of DNA consisting of 0.1 μg/mL of pSV40-βGal, 1 μg/mL of expression plasmid encoding single, double, or triple point mutations, and 0.9 μg/mL of Acta2 promoter-reporter construct, pVSMP8-luciferase^{33 (link)}. After 48 h incubation at 37°C, transfected cells were washed with PBS and harvested by lysis in 1× Passive Lysis Buffer (Promega) supplemented with protease inhibitors leupeptin, pepstatin A, and aprotinin at 1.0 μg/mL. Cleared lysates were assayed for total protein content by either Bradford or BCA™ assay (Thermo Scientific) and reporter gene expression by luciferase activity assay (Promega). Datasets were analyzed by performing a one-way analysis of variance and Tukey’s multiple comparison test with significance of p <0.05 using Prism 6 (Graphpad Software, Inc.).