Figure 5
From: CLIMP-63 is a gentamicin-binding protein that is involved in drug-induced cytotoxicity
Reducing CLIMP-63 dimer expression by siRNA transfection enhances cellular resistance to gentamicin-induced toxicity. (a) Two sets of siRNA for CLIMP-63 and scrambled control were transfected into KPT11 cells. Although the monomer form of CLIMP-63 (p63 monomer) did not show any apparent reduction, the DTT-resistant dimer form (p63 dimer) was reduced by siRNA for CLIMP-63 (p63 siRNA no. 1 and no. 2) as shown by western blotting. (b) CLIMP-63 gene knockdown by siRNA was confirmed by RT-PCR. (c) After 3 days of gentamicin treatment, cell viability was higher in CLIMP-63 siRNA-transfected cells compared with control, as determined by MTT assay (*P<0.05 between control and p63 siRNA no. 1, **P<0.01 between control and p63 siRNA no. 2). (d) Caspase-3 activity was suppressed in cells transfected by CLIMP-63 siRNA after 24 h of gentamicin treatment (*P<0.05 between control and p63 siRNA no. 1, **P<0.01 between control and p63 siRNA no. 2). Data in (c and d) are shown as mean±S.D. of three independent experiments. (e) DNA strand breaks were detected by TUNEL reaction in control siRNA-transfected KPT11 after 24 h of gentamicin treatment, but not in CLIMP-63 siRNA-transfected cells. Phalloidin labeled the filamentous actin cytoskeleton, allowing visualization of cell morphology
