Midkine partially inhibited TNFα-induced apoptosis in LNCaP cells. A. LNCaP cells in triplicate groups were not treated (as control) or treated for 4 days with 20 ng/ml TNFα, without or with 0.1 or 1 μg/ml exogenous midkine; the living cell number was counted by the trypan blue exclusion assay; the cell survival was calculated as (the living cell number of treated group ÷ the living cell number of untreated control group); the data were presented as mean ± standard deviation; *P < 0.05 compared to TNFα alone. B. LNCaP cells were transfected with the mixtures of midkine specific siRNA/Lipofectamine™ 2000, control-siRNA/Lipofectamine™ 2000, or Lipofectamine™ 2000 only (mock transfection), or no transfection as an additional control; the final concentrations used were 100 nM of siRNA or control-siRNA, and 5 μl/ml of Lipofectamine™ 2000; four h after transfection, the cells were changed into serum-free DMEM without or with 20 ng/ml TNFα; two days later, the medium supernatants were analyzed for midkine expression by Western blot. C. LNCaP cells in triplicate groups were treated as described in B; the cell survival after 2-days' treatment with 20 ng/ml TNFα was determined by the trypan blue exclusion assay as described in A; the data were presented as mean ± standard deviation; *P < 0.05 compared to the other three groups. D. LNCaP cells were treated as described in B; 16 h after treatment with 20 ng/ml TNFα, 20 μM MR-(DEVD)2 were added to the cells and incubated for 1 h, followed by addition of 1 μg/ml Hoechst 33342 for another 15 min; the red fluorescence [emitted by the cleaved MR-(DEVD)2 indicating activation of caspase-3] and blue fluorescent nuclei (stained by Hoechst 33342 to illustrate total cell number) were captured by a fluorescent microscope; original magnification: × 100.