Pa-PDT activates JNK-mediated apoptosis in R-HepG2 cells. (A) Changes in apoptosis regulatory proteins in Pa-PDT treated R-HepG2 cells. Cells (3 × 106) were treated with solvent (0.04% ethanol (CTL)) or 0.6 μM Pa-PDT, and collected at appropriate time points, where solvent control was collected at 30 min after the treatment. The cell lysates were prepared and the changes in the level of various apoptosis-related proteins were analyzed using Western blotting. The protein expression levels were semi-quantified and shown as relative intensities normalized with the band intensity of the housekeeping β-actin in each sample. Representative results from a single experiment are shown from 5 independent experiments.(B) To determinate the effect of Pa-PDT on Δψm, flow cytometry analysis was conducted in R-HepG2 cells (3 × 105/well) 1 h after light illumination (84 J/cm2, 20 min) with 0.6 μM Pa, 0.8 μM Pa, or 0.8 μM Pa with 0.5 μM JNK inhibitor. The cells were then stained with JC-1 (10 μM) for 15 min. The green and red fluorescence of JC-1 were acquired subsequently with a flow cytometer, and the results shown as mean ± SD of 3 independent experiments.