Figure 4.

The ICD markers HMGB1, CRT, and HSP70 are released as cargo of EVs shed by Cabozantinib-treated DU-145 cells. (A) Western Blot analysis of EV protein cargo (8 μg/sample) obtained from untreated (NT) or Cabozantinib-treated DU-145 (2.5 and 5 μg/ml for 48 h, respectively, 2.5CB and 5CB). As internal control, DU-145 total cell lysates (30 μg/sample) were used. Loading control was performed by Ponceau staining. The histograms represent densiometric evaluation reporting the ratio of band intensity of the sample lane vs. Ponceau lane. (B) Uptake by DCs of antigens released by untreated and Cabozantinib-treated DU-145. DU-145 cells (treated or untreated) were labeled with CSFE, and DCs were incubated with cell culture supernatants. Transfer of CSFE⁺ antigen to DCs was evaluated by flow cytometry. The negative control is the autofluorescence of DC exposed to DU-145 culture supernatant (gray histograms); the dark histograms represent the fluorescence signals derived from DCs exposed to culture supernatant of CSFE labelled DU-145. Fold increase values are the ratio between the mean fluorescence intensity (MIF) of DCs exposed to CSFE⁺DU-145 cell supernatant and DCs control.