Figure 3.

Culture in the presence of Bcc biofilms induces cell death in dHL60 cells. Bacteria (1 × 10⁶ cfu ml⁻¹) were cultured at 37°C for 0, 24, 48 or 72 h. Wells were then washed with sterile PBS and dHL60 cells (1 × 10⁷ cells ml⁻¹) were added. Discrete dHL60 cell populations were also cultured in the absence or presence of 2 ug ml⁻¹ actinomycin D or 15 μM staurosporine. Plates were incubated at 37°C for 3 h prior to harvesting of cells. Cells were lysed and assayed for caspase-3 activity (A). Columns represent the mean caspase-3 activity of triplicate dHL60 cell populations from each of three independent experiments, relative to the mean uninduced control population. Bullets indicate statistically significant differences with respect to uninduced controls (•P < 0.05, ••P < 0.01, •••P < 0.001, one-way ANOVA). Asterisks denote significant differences between biofilm age for a given species (**P < 0.01, ***P < 0.001). Cells from each population were then isolated by centrifugation and resuspended in binding buffer in the presence of annexin V-FITC and propidium iodide. Each cell population was analyzed by flow cytometry. Representative scatterplots are presented for dHL60 cells which had been cultured with (B–E) supernatants derived from planktonic or biofilm-dwelling populations of B. dolosa bacteria or (F–I) the planktonic or biofilm-dwelling B. dolosa bacteria themselves. The quantity of viable, apoptotic or necrotic cells following each culture condition is also presented for (J) supernatants and (K) planktonic or biofilm-dwelling bacteria. RNA was isolated from cells and converted to cDNA; cDNA was then analyzed semiquantitatively by qPCR (L–N). Data represent the log₂-transformed geometric means (± SE) of triplicate populations from each of three independent experiments. Bullets indicate significance differences in expression mediated by biofilms vs planktonic bacteria (•P < 0.05, ••P < 0.01, •••P < 0.001, Kruskal–Wallis test); asterisks indicate significance mediated by biofilms vs one another (*P < 0.05, **P < 0.01, ***P < 0.001).