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.