Figure 4

HIPK2 sensitized resistant cells to ADR-induced apoptosis, in hypoxia-mimicking condition. (A), RKO-C, RKO-p53i, and T98G cells were transfected with HIPK2-GFP, and the empty GFP vector. Twelve hours after transfection cells were pre-treated with CoCl₂ (200 μM) for 8 h and then treated with and ADR (2 μg/ml) for 24 h (Total time treatments: 36 h transfection, 24 h ADR; 32 h CoCl₂). Both floating and adherent cells were collected and cell viability was determined by Trypan blue exclusion. The results shown are representative of three independent experiments performed in duplicate. *p 0.001. (B), RKO-C, -p53i, and T98G cells were treated as in (A) and nuclear cell extracts analyzed by Western immunoblotting with anti-PARP antibody. The cleaved form of PARP is shown. Anti-Hsp70 was used as protein loading control. (C), RKO-C, RKO-p53i, and T98G cells transfected and treated as in (A) were analyzed by RT-PCR analysis for HIF-1α, MDR1, and Bcl2 expression. Aldolase (aldo) was used as loading control. One representative experiment from three independent experiments was shown. (D), Colony-forming ability of RKO-C, RKO-p53i, and T98G cells transfected with HIPK2-GFP and pre-treated with CoCl₂ for 22 h and ADR (2 μg/ml) for 2 h (Total time treatments: 24 h CoCl₂ and 2 h ADR). Death-resistant colonies were stained with crystal violet 10 days after transfection.