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. 2016 Apr 11;7(22):32493–32503. doi: 10.18632/oncotarget.8679

Figure 4. Overexpression of SLAMF3 in HCC cells blocks specifically MRP-1 function.

Figure 4

Cells transfected with SLAMF3 plasmid (SLAMF3+) and mock plasmid (2×106/assay) were incubated with Rhodamine (R123) at 1, 2 and 5 mM for 15 minutes and R123 fluorescence measured in a flow cytometer by gating SLAMF3+ and mock. a. Results are presented as the mean ± SD from 3 independent experiments (n = 3; * p< 0.05, **p<0.01, ***p<0.005). The MRP-1 activity was specifically measured by using eFluxx-ID® Gold multidrug resistance assay kit (NZ-51030). Unless the eFluxx-ID® dye is pumped out of the cell, the esterase cleaved dye is trapped inside the cell. Cells exhibiting drug resistance will have diminished fluorescence. Cells (mock and SLAMF3+) were incubated with Gold detection reagent with and without specific inhibitor of MRP-1 (MK-571) for 30 min in 37°C and suspended in cold PBS for flow cytometry analysis. Fluorescence of gold dye(eFluxx-ID) is measured and presented as histogramsin mock (dotted line, full) and in SLAMF3 overexpressing cells SLAMF3+ (bold line, empty) in untreated cells b. and in the presence of MRP-1 specific inhibitor MK-571 c. One representative experiment from three (n=3) is presented. The formula of calculation of multidrug resistance activity factor (MAF) as: MAFMRP= 100 × (FMRP-F0)/FMRP where FMRP corresponds to the fluorescence intensity in the presence of MRP-1 specific inhibitor MK-571 and F0 to the fluorescence intensity in absence of inhibitor. Calculated MAF is presented d. as the mean ± SD from 3 independent experiments (n = 3; **p<0.01).