Table 1.
List of antibodies used in the experiments.
Table 2.
Primers used for detection of multidrug resistance proteins.
Table 3.
Antibody concentrations used for Western blotting analysis of multidrug resistance protein expressed by endothelial cells.
Table 4.
List of control antibodies used in flow cytometry experiments.
Table 5.
Antibody concentrations used for flow cytometry analysis of multidrug resistance proteins expressed by endothelial cells.
Table 6.
Substrates and inhibitors used in functional tests.
Fig 1.
Expression of mdr proteins mRNA in endothelial cells using RT- PCR method.
The primers used for the reaction are listed in Table 2. As a control actin mRNA expression was checked. LoVo/Dx cells served as a positive control.
Table 7.
Flow cytometry analysis of multidrug resistance protein expressed by endothelial cells.
Fig 2.
Western blotting analysis of multidrug resistance protein in endothelial cells.
MDR1, MRP1 and BCRP protein levels were revealed with different sets of specific antibodies in endothelial cell extracts obtained lysing cells with RIPA buffer. Protein extracts from LoVo/Dx cells were analyzed as positive controls. MDR1/4: 180 kDa; MDR1/5: 170 kDa, MRP1/3: 180 kDa; MRP1/4: 170–220 kDa; MRP1/5: 190 kDa; BCRP/2: 72 kDa; BCRP/3 65–80 kDa and BCRP/4: 72 kDa. Equal protein loading (50 μg/line) was confirmed by β-actin expression (45 kDa).
Fig 3.
Immunocytochemical staining of endothelial cell lines on cytospin slides for mdr proteins.
LoVo/Dx cell line was used as a positive control. Slides incubated with secondary antibody (rabbit/mouse link) served as a negative control.
Fig 4.
Comparison of MDR activity factor (MAF) of endothelial cells: A) standard functional test; B) commercial functional eFluxx-ID Green test.
A) Cell lines were trypsinized, washed with medium and incubated with rhodamine 123 or calcein AM dyes. After one wash, cells were incubated in medium or medium with specific inhibitors: 10 μM of verapamil, 25 μM of MK-571 or 20 μM of novobiocin. B) Cell lines were trypsinized, washed with PBS, aliquoted and treated in triplicate with different inhibitors (20 μM of verapamil, 25 μM of MK-571, or 50 μM of novobiocin) or untreated (medium with DMSO). Tested probes (eFluxx-ID Green) were added to every sample apart from one tube (white cells). The cells were incubated with the dye in the presence or absence of inhibitors for 30 min. at 37°C.
Table 8.
Numbers indicate positive (1) or negative (0) results obtained by particular technique used. In case of ICC the gradation of results is presented as (+) and (-) score. M- mRNA; WB- Western Blotting FC- Flow Cytometry; ICC- immunocytochemistry; FA- standard functional test; FB- commercial functional test. FA* and FB* refers to functional tests applied for the whole MRP protein family. All tests were repeated at least three times, and both functional tests were each time performed in triplicate.