Fig 2: Blocking extracellular HSP (heat shock protein) 70 restricts doxorubicin-induced myocardial inflammation. A through E, At the indicated time points, the protein levels of interleukin-6, transforming growth factor (TGF)-ß1, interleukin-17A, and interleukin-10 in heart tissue homogenates were detected by Western blot analysis. In a time-dependent manner, HSP70 neutralizing antibody treatment inhibited the increase in the levels of interleukin-6 (B), TGF-ß1 (C), and interleukin-17A (D) but upregulated the level of interleukin-10 (E) in the myocardium. Representative immunoblots and the ratio of the indicated protein to GAPDH are presented. Data are the mean±SEM of 3 assays. # P<0.05, ## P<0.01, compared with normal mice; *P<0.05, **P<0.01, compared with doxorubicin-treated mice. F, The expression of the proinflammatory proteins inducible NO synthase (iNOS) and cyclooxygenase 2 as well as the anti-inflammatory protein B-cell lymphoma 3 (BCL3) in heart tissue was examined by Western blot analysis at the experimental end point. Representative immunoblots and the ratio of the indicated protein to GAPDH are presented. Data are the mean±SEM of 3 assays. # P<0.05, compared with normal mice; *P<0.05, compared with doxorubicin-treated mice.

Fig 3: BMP6 expression is upregulated via the TLR4 pathway in human monocytes.Human PBMCs were treated with sham control, LPS (100 ng/mL), or HSP70 (1 µg/mL) for 20 hours and then analyzed using the 10× Genomics platform. (A) Clustered PBMCs are displayed in UMAP format with the distribution of cells expressing BMP6. (B) Relative expression of BMP6 and the number of BMP6+ cells in each group. (C) Proportion and relative expression of the indicated genes in each cell cluster. (D) Connectivity and trajectory among cell clusters. (E) Top 20 DEGs between BMP6+ monocytes and CD14++CD16– (classical) monocytes. (F) General differentiation pathway of the monocyte lineage.

Fig 4: 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 5: 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.