Fig. 10

Modulation of A549 cell apoptosis induced by etoposide by co-cultured M0, M1 and M2 macrophages. Macrophages were co-cultured in indirect contact with A549 cells during 24 h before incubation with or without 50 μM etoposide (+/− e) during 16 h. (a) A549 cell proteins were extracted and PARP-1 and caspase-3 protein abundance was assessed by western blotting using specific antibodies. α-tubulin was used as loading control. Graphs represent the quantification of cleaved PARP-1 and cleaved caspase-3 abundance normalized by the corresponding α-tubulin in three independent experiences. Results are expressed as mean ± 1 S.D. (n = 3). (b) After the incubation with etoposide, caspase-3 and-7 activity was assayed in A549 cells by measuring the fluorescence intensity of free AFC released from the cleavage of Ac-DEVD-AFC. Results are expressed in relative caspase-3/-7 activity as mean ± 1 S.D. (n = 3). (c) After the incubation with macrophages, A549 cells were detached and stained with Annexin V-FITC and propidium iodide before fluorescence analysis by flow cytometry. The percentage of cells in the four different quadrants was calculated and the results present in different histograms where viable cells are Annexin V-/PI-, apoptotic cells Annexin V+/PI- and necrotic cells are PI +. Results are present as mean ± 1 S.D. (n = 3). Statistical analysis was carried out with the one-way ANOVA test followed by a Holm-Sidak post-test. NS: no significantly different from control cells incubated with etoposide; *, ** or ***: significantly different from control cells incubated with etoposide respectively with p < 0.05, 0.01 or 0.001