Table 1.
Percentage of Annexin V, Propidium Iodite (PI) and Monodansylcadaverine (MDC) positive HeLa cells untreated and at different recovery times after incubation with Rose Bengal Acetate 10−5 M 1 h and irradiation with 1.6 J/cm2 90 sec.
Figure 1.
Surface exposure of CRT and ERp57 in RBAc-PDT-treated HeLa cells.
A. Kinetic of CRT and ERp57 membrane translocation (ecto-CRT and ecto-ERp57) was performed by Western blot of electroblotted to nitrocellulose membrane SDS-PAGE of membrane proteins fraction (30 µg protein/lane) of RBAc-PDT treated HeLa cells (10−5 M RBAc, 1 h, 1.6 J/cm2, 90 sec) in the presence of 3-MA (10 mM) and Nec-1 (300 µ) (apoptotic cells) and in the presence of z-VAD-FMK (20 µM) and 3-MA (10 mM) (autophagic cells) at indicated time intervals. Monoclonal antibodies against CRT (63 kDa) and ERp57 (57 kDa) were used. The amount of CRT and ERp57 proteins was reported as band intensity ratio (treated/untreated) measured by densitometer analysis. E-cadherin (97 kDa) and β-actin (45 kDa) expression is shown as control. Actin is absent in the membrane protein fraction. The data are the mean ± SD of three independent experiments. The values of the apoptotic cells are always significantly different (p<0.05) with respect to autophagic cells. The values of apoptotic cells values are significantly different (p<0.05) with respect to untreated cells for both ecto-CRT and ERp57. Unless the short times after PDT (2 h) whose values are significantly different (p<0.05) with respect to untreated cells, for all the other time points, the values of ecto-CRT and ecto-ERp57 of autophagic cells are not different from untreated cells. One representative Western Blot is shown out of the three independent experiments performed. B. Fluorescence micrographs of non permeabilized (a–c) and permeabilized (a’–c’) HeLa cells immunostained for CRT: viable (a, a’); 8 h (autophagic cells) (b, b’) and 12 h (apoptotic cells) (c, c’) of recovery after RBAc-PDT. Bar = 10 µm.
Figure 2.
Involvement of ecto-CRT in phagocytosis of RBAc photosensitized HeLa cells.
Phase contrast micrographs (a, b) and fluorescence micrographs of Hoechst (1 µg/mL) labeled (a’–b’) apoptotic RBAc-PDT-induced (RBAc 10−5 M 60 min followed by 90 seconds irradiation with green light (1.6 J/cm2) and then by recovery in fresh medium for 12 h, in the presence of 3-MA 10 mM and Nec-1 300) HeLa cells co-incubated with human macrophages. a–b) inhibition of ecto-CRT by polyclonal chicken anti-CRT. Bars = 10 µm.
Table 2.
Phagocytosis index and rate, percentage of binding and number of apoptotic and autophagic HeLa cells bound per isolated human macrophages.
Figure 3.
Surface exposure of HSP70 and HSP90 in RBAc-PDT-treated HeLa cells.
Kinetic of HSP70 and HSP90 membrane translocation (ecto-HSP70 and ecto-HSP90) was performed by Western blot of electroblotted to nitrocellulose membrane SDS-PAGE of membrane proteins fraction (30 µg protein/lane) of RBAc-PDT treated HeLa cells (10−5 M RBAc, 1 h, 1.6 J/cm2, 90 sec) in the presence of 3-MA (10 mM) and Nec-1 (300 µ) (apoptotic cells) and in the presence of z-VAD-FMK (20 µM) and 3-MA (10 mM) (autophagic cells) at the indicated time intervals. Monoclonal antibodies against HSP70 (70 kDa) and HSP90 (90 kDa) were used. The amount of HSP70 and HSP90 proteins was reported as band intensity ratio (treated/untreated) measured by densitometer analysis. E-cadherin (97 kDa) and β-actin (45 kDa) expression is shown as a control. Actin is absent in the membrane proteins fraction. The data are the mean ± SD of three independent experiments. The values for ecto-HSP70 of the apoptotic cells are always significantly different (p<0.05) with respect to autophagic cells. The values of apoptotic and autophagic cells values are significantly different (p<0.05) with respect to untreated cells for both ecto-HSP70 and ecto-HSP90. One representative Western Blot is shown out of the three independent experiments performed.
Figure 4.
RBAc-PDT induces an early release of HSP70 and HSP90 and a late and passive release of HMGB1 in HeLa cells.
Kinetic of released HSP70 and HSP90 was performed by Western blot of electroblotted to nitrocellulose membrane SDS-PAGE of the conditioned media proteins (30 µg protein/lane) of RBAc-PDT treated HeLa cells (10−5 M RBAc, 1 h, 1.6 J/cm2, 90 sec) in the presence of 3-MA (10 mM) and Nec-1 (300 µ) (apoptotic cells) and in the presence of z-VAD-FMK (20 µM) and 3-MA (10 mM) (autophagic cells) at indicated time intervals. Monoclonal antibodies against HSP70 (70 kDa), HSP90 (90 kDa) and HMGB1 (29 kDa) were used. The amount of HSP70, HSP90 and HMGB1 proteins was reported as band intensity ratio (treated/untreated) measured by densitometer analysis. Actin is absent in the conditioned media proteins. The data are the mean ± SD of three independent experiments. The values (4–12 h) of ecto-HSP70 of apoptotic cells are significantly different (p<0.05) with respect to autophagic cells. HSP90 and HMGB1values of apoptotic cells were always significantly different (p<0.05) with respect to autophagic cells. HSP70, HSP90 and HMGB1 values of apoptotic and autophagic cells were always significantly different (p<0.05) with respect to untreated cells. One representative Western Blot is shown out of the three independent experiments performed.
Figure 5.
RBAc-PDT induces release of ATP in HeLa cells.
Kinetic of intracellular and extracellular released ATP was performed by ELISA (A) and luciferin-luciferase based (B) assay on the conditioned media of RBAc-PDT treated HeLa cells (10−5 M RBAc, 1 h, 1.6 J/cm2, 90 sec) in the presence of 3-MA (10 mM) and Nec-1 (300 µ) (apoptotic cells) and in the presence of z-VAD-FMK (20 µM) and 3-MA (10 mM) (autophagic cells) at indicated time intervals. The amount of ATP, reported as ng/mL or as concentration (M), is the mean ± SD of three independent experiments. The values of extracellular ATP both of apoptotic and autophagic cells are significantly different (p<0.05) with respect to untreated cells. Extracelluar ATP values of apoptotic cells were always significantly different (p<0.05) with respect to autophagic cells. Intracellular ATP values of apoptoticc cells were always significantly different (p<0.05) with respect to autophagic and untreated cells.