Figure 4
Levels of CD271 expression modulate drug response. (a) Left panels: IF microscopy of MeWoFote cells stably transfected with a validated CD271-targeting shRNA (shCD271_5-2, clone 5-2) or control (shCtl.) for CD271 (red, left panels), phalloidin (red, center panels), presence of Ki67 (green, right panels) or bright field is shown. Numbers represent % of Ki67-positive cells±s.d. of n=4 counts related to 4′,6-diamidin-2-phenylindol (DAPI) that served as nuclear stain, scale bars indicate 50 μm. Right panel: IF microscopy of clone 5-1 (shCD271_5-1) for phalloidin or bright field is shown. (b) Left and right panels: determination of cell viability and apoptosis of MeWoFote cells, clone 5-2/clone 5-1 modified as described in a following a dose-dependent treatment with fotemustine (μg/ml) for 48 h. Viability is indicated in %±s.d. of n=8 replicates, related to untreated (100% viability) cells, a representative out of three biological replicates is shown, UT=untreated; 0=solvent control. Apoptosis was determined by an ELISA-based measurement of apoptosis induced DNA-fragmentation of clone 5-2 and clone 5-1 after 48 h, scales indicate apoptosis as OD405 values, *P⩽0.05; **P⩽0.01; ***P⩽0.001. (c) Left panels: IF microscopy of MeWoVind cells following stable knock-down of CD271 by a validated shRNA (sh#4) for levels of CD271 (red), Ki67 (green) or bright field. Center and right panel: determination of cell viability CD271 knock-down cells (pool) following a dose-dependent treatment with vindensine and fotemustine for 48 h. Representative experiments out of n=3 are shown.
