Effects of antisense PONs on P-gp localization and actin association on CEM-VBL100 cells.

(A) Immunofluorescence analysis of P-gp cellular distribution in ERM sense (left panel) and antisense (right panel) PON-treated CEM-VBL100 cells. Arrows (left panel) point to the polarized distribution of P-gp in sense PON–treated cells; in contrast, antisense PON–treated cells (right panel) did not show any feature of polarization, together with an altered P-gp distribution on the cell surface. Bar = 5 μm. (B) Immuno-SEM analysis of P-gp distribution in ERM sense (upper panels) and antisense (lower panels) PON-treated CEM-VBL100 cells. The polarized morphology of sense PON–treated CEM-VBL100 cells (arrows, upper left panel) was completely lost after antisense PON treatment (arrows, lower left panel). Consistently, P-gp distribution, highly polarized on uropoidal formations (arrows, upper right panel), was clearly altered and unpolarized in antisense PON–treated cells (arrows, lower right panel). No appreciable labeling was detected in control conjugate samples incubated with irrelevant mAb. Bar = 5 μm. In both (A) and (B), 1 of 5 representative experiments is shown. (C) Coimmunoprecipitation analysis of CEM-VBL100 cells pretreated with sense (1) or antisense (2) ERM PONs. Actin immunoprecipitates were separated by SDS-PAGE in nonreducing (upper panel) or reducing (lower panel) conditions (see “Materials and methods”) and blotted with anti-actin or anti–P-gp mAb, respectively.