Fig. 1: Ursodeoxycholic acid induces EMT in A2780 cells.

A A2780 cells (8 × 10⁴/well) were plated to 6 well plates and were treated with 300 nM UDCA or DMSO for 48 h. Cells were scraped, RNA was prepared and the expression of the indicated gene was assessed by RT-qPCR. Three biological replicates are reported. B A2780 cells (5 × 10⁵) were plated into 10 cm Petri dish and were treated with 300 nM UDCA/DMSO for 48 h. Cells were scraped, 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. Three biological replicates are reported. C A2780 cells (2 × 10⁴ or 5 × 10⁴) were plated into 8W10E ECIS plates (3–4 well/condition). After reaching confluency, cells were treated with 300 nM UDCA or DMSO as vehicle control. Changes to resistance was measured and calculated as described in the “Materials and methods”. Three biological replicates are reported. D A2780 cells (4 × 10⁴/well) were plated into the upper chamber of a Boyden chamber and were exposed to the chemoattractant for 24 h. The chamber was dismantled and the cells on the lower surface of the membrane were DAPI stained and counted. The invasion index was calculated and was expressed as fold change compared to the control. One biological replicate is reported. The scale bar equals 200 µm. E A2780 cells (5 × 10⁴ cells/well) were plated in 96-well plates and confluent cultures were generated. Scarring was performed using TECAN Freedom Evo robot. After scarring UDCA (300 nM)/DMSO (vehicle) treatment was initiated. Cell migration to the void area was monitored using the PE Opera Phoenix instrument for 48 h. Images were analyzed using the Image J software. The % of the original (T0) void area was expressed. The scale bar equals 500 µm. The assay was performed in quadruplicate; one biological replicate is presented. The dashed line represents the initial void area. F, G A2780 cells (10³ cells/well) were plated to 96 well plates and were treated with UDCA in the indicated concentrations for 48 h. On F concentrations correspond to the serum reference concentrations of UDCA (red line), on G concentrations correspond to the serum therapeutic concentrations of UDCA (blue line). Cell numbers were determined using the SRB assay. Treatments were performed in duplicates. On F 6, on G 8 biological replicates are reported. Data are represented as average ± SD with the exception of D, where no SD is presented. On F, G values were normalized for vehicle-treated cells, absorbance for vehicle-treated cells equals to 1. Normality was assessed using the Shapiro–Wilk test. Values were compared using unpaired t-test, except for (F, G). On F, G one-way ANOVA was performed, in the post-hoc test all values were compared to the vehicle control. * represent statistically significant difference between vehicle and UDCA-treated groups at p < 0.05. On D, E brightness and contrast were adjusted. CTL control, DMSO dimethyl-sulfoxide, n.s. not significant; TCF7L2 transcription factor 7-like 2, UDCA ursodeoxycholic acid.