Fig. 4.

Gentamicin-induced up-regulation of Hrk is mediated by MAPKs. (a) Caspase-3 activation experiments indicate that gentamicin-induced apoptosis of HEI-OC1 cells is prevented by preincubation with LCAR and ERK1/2 inhibition, but enhanced by inhibition of JNK. JNK inhibition also interferes with the preventive effect of LCAR. Values are normalized (control = 100%). (b) RT-PCR results confirmed that the effects of the inhibitors of MAPK on caspase-3 activation were associated with changes in Hrk expression. (c) Phosphorylation studies indicate that gentamicin activates ERK1/2 and inactivates JNK. LCAR, in turn, is able to reverse the gentamicin-induced inactivation of JNK. (d-f) Confocal microscopy of HEI-OC1 cells triple-labeled with gentamicin (green), anti-p-ERK (red), and DAPI (blue) confirms that gentamicin activates ERK and demonstrates that gentamicin also induces its translocation to the nucleus. A cell with numerous gentamicin-filled vesicles in the cytoplasm and p-ERK concentrated in the nucleus is pointed out with an arrow. An arrowhead indicates a cell that incorporates only a small amount of gentamicin and shows cytoplasmic labeling with anti-p-ERK.