Fig 1: ABCC4 protein expression level in CRC cell lines. Western blot performed in standard reducing SDS PAGE conditions using goat anti ABCC4 (#PA5 18315, Thermo Scientific) and rabbit anti ABCG2 (#ORB 155559 Biorbyt). (A) Protein expression level of ABCC4 and ABCG2 in HT-29 stably overexpressing transcription factor Snail (HT29/Snail) and control HT-29. ABCC4 level in the membrane fraction (obtained by biotynylation using EZ-Link Sulfo -NHS-Biotin Thermo Scientific kit) of HT-29 control cells and HT-29 Snail n = 3. (B) ABCC4 protein expression level in CRC cells in different states of EMT: CCD841CoN (most epithelial), CaCo-2 (moderate EMT), and Colo-320 (most mesenchymal) n = 3. (C) ABCC4 protein abundance in Extracellular Vesicles (EVs) released from HT-29 control cells and two HT-29 stably overexpressing transcription factor Snail clones (HT-29/Snail and HT-29/Snail17), n = 2. (D) Intracellular cAMP level measurement. Accumulation of cAMP in HT29 cells was measured using a cAMP competitive kit (#581001 Cayman Chemical). Cells were incubated for 24 h with MK571 20 µM, or untreated ones were assayed according to the manufacturer’s protocol. Calculation were conducted using the Cayman data sheet. cAMP concentration of HT29 was set as 100%. T-test performed, n = 5; * p < 0.05; ** p < 0.005; *** p < 0.001. NS—not statistically significant. (E) PKA phosphorylation profile analysis. HT29 Snail cells were seeded on a 6-well plate. Then, 24 h after, full growth medium was changed into starving (FBS free) medium for 24 h. Next, 20uM of MK571 was added to cells for 60, 30, 5, and 1 min. Cells without the starving procedure were used as a positive control, and negative control cells were not treated with MK571. Phosphorylation profile analysis was performed using phospho-(ser/thr) PKA Substrate Antibody #9621 (Cell Signaling Technology). Significant time- (exposure) related impact on the phosphorylation profile was observed for 42 kDa and 95–100 kDa proteins in HT29 Snail cells compared to no time-related changes in control cells, n = 3. (F) HT-29/Snail PKA phosphorylation profile analyzed with densitometry; statistical significance estimated using T-test. * p < 0.05; ** p < 0.005; *** p < 0.001. NS—not statistically significant.