Fig. 3: Ursodeoxycholic acid induces NRF2 expression and reduces oxidative stress, but does not modulate cell proliferation.
A A2780 cells (105/well) were plated in 6-well plates and were treated with UDCA or DMSO as vehicle for 48 h. Cells were scraped, RNA was prepared and the expression of Nrf2 gene was assessed by RT-qPCR. Three biological replicates are reported. B–D A2780 cells were plated in 6 cm petri dishes (106 cells/dish) and were treated with 300 nM UDCA or DMSO as vehicle for 48 h. Total protein was prepared and lysates were subjected to SDS-PAGE and Western blot. Blots were developed with the antibodies indicated and were evaluated by densitometry. Number of biological replicates on B is 2 for the Novus and 2 for the Abcam antibody; on C, the number of biological replicates is 4; on D, the number of biological replicates is 3. E A2780 cells were plated in 24 well plates (104 cells/well) and were treated with 300 nM UDCA or DMSO as vehicle control for 48 h. Cells were stained with 2.5 µM Hydroethidine for 30 min and fluorescence signal was measured by flow cytometry. Treatments were performed in 6 replicates. Result from 2 independent experiments is reported. All values are depicted as mean ± SD. The number of biological replicates is the following: B Abcam n = 2, Novus n = 2, C n = 4, D n = 3. Normality was assessed using the Shapiro–Wilk test. Densitometric values were compared using unpaired t-test. ** and *** indicate statistical difference between vehicle and UDCA-treated cells at p < 0.01 or p < 0.001, respectively. DMSO dimethyl sulfoxide, HE hydroethidine, KEAP1 Kelch-like ECH-associated protein 1, NRF2 nuclear factor erythroid 2-related factor 2, UDCA ursodeoxycholic acid, 4HNE 4-hydroxy-nonenal.
