Erythrosine-Based PDT of Oral Epithelial Cells Figure 3. Erythrosine co-localization analysis. Sub-cellular co-localization of erythrosine in both DOK and H357 cells. (A and B) LysoTracker localization (for lysosomal visualization) and MitoTracker localization (for mitochondrial visualization) was compared with 1136.50 mM erythrosine localization in both DOK (A) as well as H357 (B) cells. Also, co-localization frequency scatter plots, Pearson’s coefficient (r) and Mander’s coefficient (M) were calculated to evaluate the overlap between LysoTracker/MitoTracker localizations and erythrosine localizations. (C) Co-localization frequency colour map indicates the estimation of co-localizations on the basis of ‘heat map schemes’ such that hotter colours tend to indicate high co- localization. Thus, red/maroon indicates 100% co-localization, yellow-green indicates 50% co-localization and purple/dark blue indicates 0% co- localization. doi:10.1371/journal.pone.0034475.g003 with highly depolarized mitochondria shifted towards normalcy. However, at 24 hrs, the DYm had collapsed further (Figure 4A). Low dose-treated H357 cells showed no appreciable loss of their overall DYm at the earlier recover time-points following PDT (Figure 4B). However, similar to the DOK cells (Figure 4A), at 24 hrs the DYm suddenly collapsed drastically (Figure 4B). Similarly, DOK and H357 cells treated with high dose (1136.5 mM–122.58 J/cm2) also showed different responses ini- tially. While DOK cells immediately lost their DYm after PDT (Figure 4A), the H357 cells initially resisted the change in DYm at the earlier recovery time-points after PDT (Figure 4B). However, at 24 hrs after PDT, the DYm collapsed drastically for both cell lines thereby pointing towards severe mitochondrial outer- membrane permeabilization (MMP). Cell Death Morphology analysis using CLSM and SEM Untreated (Control) DOK and H357 cells showed a normal morphology in both the SEM (Figure 5 and 6) and CLSM analyses (Figure 7). Following PDT with low dose erythrosine-PDT (71.03 mM–122.58 J/cm2), we observed apoptotic-like character- istics such as membrane blebbing, cell shrinkage, loss of extensive cell-cell linkage and formation of apoptotic bodies at early recovery time points post-PDT (0–4 h) followed by secondary necrosis at 24 h post-PDT; in both DOK (Figure 5 and 7) and H357 cells (Figure 6 and 7). Following PDT with high dose erythrosine-PDT (1136.5 mM–122.58 J/cm2) we observed prom- inent necrotic characteristics such as extensive membrane and cell disintegration, extensive karyolysis and the presence of large amounts of cellular debris at all recovery time point post-PDT in DOK cells (Figure 5 and 7) along with some apoptotic characteristics in H357 cells (Figure 6 and 7). Caspase Activity Assay Untreated (Control) DOK and H357 cells showed a constant, basal caspase 3/7 activity at all recovery time points. But after a low dose of erythrosine-PDT (71.03 mM–122.58 J/cm2), we observed an increase in the caspase 3/7 activity in both the cell lines (Figure 8A and 8B). The caspase 3/7 activity initially PLoS ONE | www.plosone.org 5 April 2012 | Volume 7 | Issue 4 | e34475