Fig. 1. Cyclophilin B is transcriptionally upregulated by ER stress. H9C2 cells were treated with 1 µM Tg and 10 µg/ml Tm under proliferation conditions (PM) for 48 hours or under differentiation conditions (DM) for 24 hours. Controls (con) are untreated cells. (A) Western blot analysis with ER stress marker antibodies to Bip, Grp94 and PDI. CypB protein level was quantified by densitometry. Actin was utilized as a loading control. Numbers under each band of CypB are representative of at least three different experiments and are expressed as the means ± s.d. ^*P<0.05 versus untreated cell in PM; ^#P<0.05, versus untreated cell in DM. (B) Micrographs of ER stress-induced cell death. (C) Western blot with antibodies to apoptosis markers: caspase 3 (cleaved form), Bax (mitochondrial), procaspase 12 and cytochrome c (cytosolic). When exposed to Tg or Tm, cleavage of caspase 3, reduction of procaspase 12, cytochrome c release and Bax translocation were observed. β-tubulin and HSP60 were used as a loading control for the cytosolic and mitochondrial fractions, respectively. Numbers under each band of procaspase 12 are representative of at least three different experiments and are expressed as the mean ± s.d. ^*P<0.05 versus untreated cell in PM; ^#P<0.05, versus untreated cell in DM. (D) Measurement of apoptosis-induced chromatin condensation by Hoechst 33342 staining. Yellow arrows indicate apoptosis-induced chromatin condensation and fragmentation. In the bar graph the data are expressed as the means ± s.d. obtained from five independent experiments. ^*P<0.05 versus untreated cells in PM condition; ^#P<0.05 versus untreated cell in DM condition. (E,F) Semi-quantitative RT-PCR analysis of CypB transcripts. GAPDH mRNA amplification was used as a control for RT-PCR. Transcript levels of CypB in untreated cells were used as a reference. Numbers under the each band of CypB are representative of at least three different experiments and are expressed as the means ± s.d. ^*P<0.05 versus untreated cell in PM; ^#P<0.05, versus untreated cell in DM (E). Actinomycin D (AD) was employed to inhibit mRNA synthesis. ^*P<0.05 actinomycin D-treated cell versus untreated cell (F).