Fig. 2

Exosomes from breast cancer cells exacerbate DOX-induced cardiomyocyte pyroptosis in vitro. a EXOs from BCCs were characterized using transmission electron microscopy (TEM). Scale bar: 100 nm. b Particle size distribution was determined using NanoSight tracking analysis. (NTA). EXO particle concentration in the cell culture supernatant with or without DOX. c Western blot analysis of HSP70, CD81, TSG101, GM130, and Calnexin in cell lysate and 4T1-derived EXOs with or without DOX. d In vitro and ex vivo breast cancer EXO uptake analysis showing fluorescence-labeled EXOs internalized by human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) or adult murine ventricular cardiomyocytes (AMVCs) observed under a confocal microscope. For in vitro analysis, PKH67-labeled EXOs were harvested and subsequently incubated with hiPSC-CMs. For ex vivo analysis, AMVCs were isolated from mouse hearts 24 hours after tail vein injection of PKH67-labeled EXOs. Scale bar: 20 μm. e, f Representative images of AMVCs and hiPSC-CMs after DOX + N-BCC-EXO or DOX + D-BCC-EXO treatment for 24 hours and (g, h) assessments of cell viability, (i, j) CASP1 expression levels, and (k, l) LDH release levels (n = 5) are shown. Top row: bright-field images, scale bar: 50μm; middle row: CASP1 immunofluorescence (green), scale bar: 20 μm; bottom row: merged images of α-actinin (red), CASP1 (green), and DAPI (blue), scale bar: 20 μm. m Pyroptosis-related protein levels in AMVCs were assessed via Western blotting. All the experimental groups were compared with the DOX group via one-way ANOVA. n IL-18 levels in AMVCs were determined using a colorimetric method (n = 5). “N-BCC-EXOs” denotes normal breast cancer cell EXOs, and “D-BCC-EXOs” denotes DOX-induced breast cancer cell EXOs. Red represents α-actinin, green represents CASP1, and blue represents DAPI. “DOX” indicates doxorubicin, and “ns” indicates non-significant. Data are presented as Mean ± SD