Fig 2: Anti-cancer activities of HSP90 inhibition in TNBC cells. a Differential sensitivity of ganetespib in TNBC cell lines. A panel of seven TNBC cell lines (HS578T, MDA-MB-453, MDA-MB-468, MDA-MB-231, HCC 1937, BT20 and HCC1143) were treated with increasing concentrations of ganetespib for 72 h and subjected to resazurin-based cell viability assay. Cell viability (%) for each treatment was expressed relative to the DMSO-treated control. Representative graph from at least three independent experiments each performed in triplicate and error bars indicate SEM. IC50 (the drug concentration that reduces cell viability to 50% of control) was calculated from the three independent experiments for each cell line. b Downregulation of HSP90 client proteins and induction of apoptosis by ganetespib. HS578T, MDA-MB-231 and HCC1143 cell lines were treated with increasing concentrations of ganetespib (10, 30 and 100 nM) for 24 h. Protein lysates of the treated cells were subjected to western blotting analysis and blotted with the indicated antibodies. Cleaved PARP was used to assess apoptosis and Hsp70 was used as a pharmacological marker for inhibition of Hsp90 activity. PPIB was used as loading control. c Generation of HSP90i-resistant clones. Hs578T cells (105) were plated and treated with 30 nM of ganetespib. After 6 weeks, colonies started to form and two independent clones (CR2 and CR3) were picked from different plates

Fig 3: Immunoblot analysis of the whole cell lysates, and the exosome and MV lysates collected from MDA-MB-231 cellsLysates of MDA-MB-231 cells (WCL), as well of the exosomes and MVs these cells generate, were incubated with antibodies to detect the expression levels of the general EV markers Hsp90 and Flotillin-2, the exosome marker CD63, the MV marker Annexin, and the WCL marker IκBα.

Fig 4: Anlo increased the expression of PDL1 by ROS/JNK/AP-1 pathway in vitro. (a) ROS production was determined and analyzed by flow cytometry. (b) Representative western blots of HSP90, T-JNK, P-JNK, P-C-FOS, P-C-JUN, IFN-a, IFN-ß, and IFN-?. (c) The expression of mRNA levels of IFN-a, IFN-ß, and IFN-?. (d) RNA sequence data analysis of CT26 tumor.

Fig 5: ATV-Exos activated angiogenesis-related AKT/eNOS pathway. HUVECs were treated with LG, HG, HG + Exos, and HG + ATV-Exos, and the protein levels of p-AKT, AKT, p-eNOS, eNOS and PTEN and HSP90 were explored by Western blotting with HSP90 being used as a control. *P < 0.05