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. 2015 Feb 18;6(9):6776–6793. doi: 10.18632/oncotarget.2882

Figure 6. CAXII is physically associated with Pgp and increases Pgp activity in chemoresistant cells.

Figure 6

(A) Confocal microscope analysis of HT29 and HT29/dx cells stained for CAXII and Pgp. The samples were analyzed by laser scanning confocal microscope for green (CAXII) or red (Pgp) fluorescence signal. Magnification: 60 × objective; 10 × ocular lens. Bar = 20 μm. (B) Biotinylated plasma membrane-derived extracts from HT29 and HT29/dx cells were immunoprecipitated (IP) with anti-Pgp or anti-MRP1 antibodies, then immunoblotted (IB) with anti-CAXII, anti-Pgp or anti-MRP1 antibodies. no Ab: samples immunoprecipitated without antibody. The figure is representative of three experiments with similar results. (C) HT29/dx cells were cultured for 48 h with fresh medium (CTRL), treated with a non targeting scrambled siRNA (scr) or with a CAXII-targeting specific siRNA pool (siCAXII). The Pgp ATPase activity was measured spectrophotometrically on Pgp-rich vesicles extracted from membrane fractions. Data are presented as means ± SD (n = 4). Versus CTRL: *p < 0.001. (D) Cells treated as reported in C were incubated for 24 h with 5 μmol/L doxorubicin, then the intracellular drug content was measured fluorimetrically. HT29 were included as control of chemosensitive cells. Data are presented as means ± SD (n = 4). Versus HT29 CTRL: *p < 0.001; versus HT29/dx CTRL: ° p < 0.001. (E) Cells treated as reported in C were grown for 72 h in fresh medium (CTRL), or in medium containing 5 μmol/L doxorubicin (DOX) or 1 μM irinotecan (CPT11), then stained with neutral red dye. The absorbance of viable cells was measured spectrophotometrically. Data are presented as means ± SD (n = 4). For HT29 and HT29/dx cells, versus CTRL: *p < 0.001; for HT29/dx cells, versus wild type HT29/dx DOX or HT29/dx CPT11, respectively: ° p < 0.002. (F) Biotinylated plasma membrane-derived extracts from human chemosensitive lung cancer A549 cell and chemoresistant A549/dx cells, human chemosensitive osteosarcoma U2-OS cells and chemoresistant U2-OS/dx580 cells were immunoprecipitated (IP) with an anti-Pgp antibody, then immunoblotted (IB) with an anti-CAXII or an anti-Pgp antibody. no Ab: samples immunoprecipitated without antibody. The figure is representative of two experiments with similar results. (G) A549/dx and U2-OS/dx580 cells were grown in fresh medium (CTRL), treated with a non targeting scrambled siRNA (scr) or with a CAXII-targeting specific siRNA pool (siCAXII). The Pgp ATPase activity was measured spectrophotometrically on Pgp-rich vesicles extracted from membrane fractions. Data are presented as means ± SD (n = 3). For both cell lines, versus CTRL: *p < 0.001.