Fig 2: Quantification of ATP and HSP70 release from tumor cells after NB-PDT. ATP in the supernatant was detected 4 h after NB-PDT via a luminescence assay and graphs show luminescence values relative to untreated cells for (a), A431 cells and (b), scc-U8 cells. Additionally, released HSP70 was detected 24 h after treatment using ELISA on (c), A431 cells and (d), scc-U8 cells. NT, untreated; LD50, mild cytotoxic NB-PDT; LD100, highly cytotoxic NB-PDT; Light, only light control; NB-PS, only nanobody-photosensitizer (NB-PS) conjugate control. Significance is displayed as * p = 0.05 and ** p = 0.01.

Fig 3: Cellular localization of heat shock protein 70 (HSP70) and high mobility group box 1 protein (HMGB1) on tumor cells treated with NB-PDT. Tumor cells were left untreated (NT) or treated with NB-PDT using 7D12-PS (LD50 or LD100), and 4 h later stained for HSP70 or HMGB1. Staining of HSP70 (red) was performed on non-permeabilized (a), A431 cells and (b), scc-U8 cells. Intracellular staining of HMGB1 (green) was performed on (c), A431 cells and (d), scc-U8 cells. Cell nuclei were additionally stained with DAPI (blue). Top panels depict only the damage-associated molecular pattern (DAMP) signal, while merged images are shown on the bottom panels. Scale bar, 20 µm.