Figure 4.
4-1BB triggering enhances survival of kidney epithelium in vivo. (A) Kidney epithelial cells from 4-1BB KO BALB/c or Rag2γc KO mice were cultured in vitro for 7 days and treated with 2 μg/ml cisplatin or PBS for 24hrs. RT-PCR was performed with cDNA of the cultured cells to determine 4-1BB transcripts. (B) Rag2γc KO mice were injected i.p. with indicated doses of cisplatin and also single-injected with 100μg of rat IgG or anti-4-1BB mAb. Complementary DNA was synthesized with total RNA from kidney 72 hrs after cisplatin injection. Real-time PCR (I) and RT-PCR (II) were performed to assess Bcl-2, Bfl-1, and Bax expression as describe above. Relative gene expression was normalized to GAPDH and shown as the fold increase compared to that of rat IgG-treated group. (C, D) Kidney epithelial cells from Rag2γc DKO or 4-1BB KO mice were transfected with either gene-specific siRNA against Bfl-1, Bax or nontargeting control siRNA according to manufacturer’s instruction (Santa Cruz Biotechology). (C) Decrease in Bfl-1 or Bax mRNA upon corresponding siRNA transfection was determined by RT-PCR. (D) Cells were treated with 2 μg/ml cisplatin and/or 10 μg/ml of rat IgG or anti-4-1BB, and the absolute numbers of viable cells were determined at the indicated time points. Each group contained three mice and the results are representative of three independent experiments. All plotted data are means ± SD (*, P < 0.05; **, P < 0.01; ***, P < 0.001).