Induction of caspase 3 and TG2- dependent apoptosis by ACR in JHH-7 cell cultures. A, JHH-7 and HC cells were seeded in 35 mm dishes containing glass coverslips at 2 × 105/dish, and treated with 10 μM ACR or vehicle (0.1% ethanol) for 24 h. Cells were fixed and stained combination with Hoechst 33258, and TUNEL (left panels) or anti-CLSp1 antibody (right panel). Scale bar, 50 μm. B, JHH-7, HuH-7, HepG2 and HelaS3 were seeded at 1 × 104 cells/96 well microplates and treated with the indicated concentrations of ACR or vehicle for 24 h. Viable cell counts are plotted as percentages of each control culture treated with vehicle. C, JHH-7 cells were seeded as before and treated either with the indicated concentrations of ACR for 48 h, or with 10 μM ACR for the indicated times. Cells were fixed and stained with Hoechst 33258, TUNEL, and anti-TG2 antibody. The numbers of total and apoptotic cells with TUNEL or TG2 positivity in each dish were counted and plotted. D, JHH-7 cells were seeded as before and transfected with either pSG5 or anti-sense (AS) TG2-pSG5. The next day cells were treated with either vehicle or 10 μM ACR for 24 h in the presence or absence of 100 μM zDEVD-fmk, with 1 mM 5-(biotinamido)-pentylamine being included during the last 2 h incubation. Cells were fixed and stained with Hoechst 33258 (upper panels), TRITC-conjugated streptavidin (middle panels), and TUNEL (bottom panels). Arrowheads indicate apoptotic cells with chromatin condensation. Scale bar, 50 μm. E, JHH-7 cells were treated as in (C). The numbers of total and apoptotic cells with TUNEL (green colors) or TG2 (orange colors) positivity in each dish were counted and plotted as bar graphs. Their percentages relative to total cell number are given on the right hand-side of each bar graph. Panels A-E show representative results from 3 different experiments with similar results.