Ji-Young Kim, et al. gondii and freshly isolated cells showed apoptosis of 43.6%, 37.0%, and 15.8%, respectively after incubation for 5 h (data not shown). The quantity of apoptotic spleen cells were 80.3%, 30.1%, and 23.8%, respectively, after incubation for 18 h (Fig. 2, A, D, F). Similar results were found upon repeated experiments. Levels of Bax mRNA and protein expression in spleen cells were reduced upon treatment with T. gondii RT-PCR analysis of Bcl-2 family genes was performed on mouse spleen cells incubated with AD, or AD/T. gondii for 30 min, 60 min or 120 min at 37 . Expression of pro-apoptotic Bax mRNA was down regulated in AD/T. gondii infected spleen cells (Fig. 3). Western blot an- alysis showed that the production of Bax protein was decreased in spleen cells treated with AD and infected with T. gondii, while Bak protein levels remained unchanged (Fig. 4). Expression levels of anti-apoptotic Bcl-2 and Bcl-XL mRNAs remained constant in spleen cells treated with AD and in- fected with T. gondii (Fig. 3). Spleen cells infected with T. gondii showed indistinct Bax and Bcl-2 mRNA levels. Moreover, Bcl-2 protein did not appear in AD/T. gondii infected cells. Caspase 3 and PARP protein activation was suppressed in T. gondii infected spleen cells Activation of caspase 3 and PARP proteins was (min) Bax Bcl-2 Bcl-XL β-actin Fig. 3. RT-PCR analysis of Bcl-2 family genes in mouse spleen cells. Expression of pro-apoptotic Bax mRNA was down regulated and levels of anti-apoptotic Bcl-2 and Bcl-XL mRNA remained constant in spleen cells treated with AD/T. gondii. Spleen cells were co-cultured with either actinomycin-D and/or T. gondii for 30 min, 60 min, or 120 min followed by extraction of total cellular RNA.. (M): marker; (-): negative; (AD): actinomycin-D (5 μg/ mL); (AD + T.g): actinomycin-D + T. gondii infection. Yonsei Med J Vol. 47, No. 6, 2006 suppressed in spleen cells treated with AD and infected with T. gondii. Down regulated, cleaved caspase 3 at 120 min post infection and reduced production of PARP in T. gondii infected cells were detected by Western blotting (Fig. 4). Although cleaved caspase 8 was observed in spleen cells treated with AD and infected with T. gondii, it was not expressed in spleen cells treated with AD only (Fig. 4). Activation of NF-κB in spleen cells infected with T. gondii We examined the role of NF-κB in the anti- apoptotic response of T. gondii infected spleen cells by EMSA and Western blotting. NF-κB activity was apparent at 120 min in spleen cells treated with AD and infected with T. gondii, which decreased by 180 min (Fig. 5A). T. gondii infected spleen cells showed more activated NF-kB than uninfected cells at 60 min on EMSA. A similar pattern of NF-κB activity in total cell and nuclear extracts was observed by Western blot (Fig. 5B). Decreased IκBα was associated with increased NF- κB activity. Fig. 4. Western blot analysis of Bcl-2 family proteins, caspase 3, caspase 8 and PARP in mouse spleen cells in the presence or absence of T. gondii and actinomycin-D after incubation for 0 min, 60 min or 120 min. Production of Bax protein was decreased in spleen cells treated with AD/T. gondii, while levels of Bak protein was unchanged. Down regulation of cleaved caspase 3 at 120 min post infection and reduced production of PARP in T. gondii infected cells was detected by Western blotting. (AD): actinomycin-D (5 μg/mL); (AD + T.g): actinomycin-D + T. gondii infection.