Thbs1

Hearts were collected at the endpoint (after 72 hours of IR, and controls after ultrasound data were collected) from experimental animals after KCl injection (40 mM). Hearts were thoroughly washed in PBS. For fixation, hearts were perfused with 4% paraformaldehyde and preserved in Peel‐A‐Way disposable plastic tissue embedding molds (Polysciences Inc) filled with tissue freezing media (Triangle Biomedical Sciences) and stored at −70°C until analysis. Tissue sections (5 µm in thickness) were cut using a Leica CM 3050S Cryostat. Sections were placed on Superfrost plus glass slides, air‐dried, and processed for histological and immunohistochemical (IHC) staining using a standard IHC protocol. Primary antibodies applied overnight included anti‐Drp‐1 and Thbs‐1 (all from Abcam). Secondary antibodies labeled with Alexa Fluor 488 and Texas Red (Invitrogen) were applied for immunodetection of these proteins. Stained slides were imaged for fluorescence at 100× magnification using an Olympus FluoView 1000 laser scanning confocal microscope. The total fluorescence (green or red) intensity in five random fields (for each experimental sample) was measured with Nikon Elements image analysis software. Fluorescence intensity values for each experimental group were averaged and presented as mean fluorescent intensity (MFI).

Multiplexed immunofluorescence (mIF) was performed by staining 4‐μm‐thick formalin‐fixed, paraffin‐embedded whole tissue sections with standard, primary antibodies sequentially and paired with a TSA 7‐color kit (D110071‐50T, Yuanxi Bio). The primary antibodies were MUC1 (Abcam; catalog no. ab109185; RRID:AB_10862483), THBS1 (Abcam; catalog no. ab267388), FGF7 (Sabbiotech; catalog no. 31162‐1), MS4A4A (Abcam; catalog no. ab271069), ZEB1 (Proteintech; catalog no. 21544‐1‐AP; RRID:AB_10734325), VEGFC (Proteintech; catalog no. 22601‐1‐AP; RRID:AB_2879132), and CD68 (Abcam; catalog no. ab213363; RRID:AB_2801637). After multiplexed immunofluorescence staining, each slide was treated with two drops of DAPI, washed in distilled water, and manually coverslipped. Slides were air‐dried and mounted with anti‐fade mounting medium, and pictures were taken with PANNORAMIC MIDI II. Images were analyzed using Indica Halo software.

SK-MEL-28 cells were seeded in 60 mm culture dishes at a density of 6 × 10⁵ cells/dish with 240 µg/ml HANPs and without HANPs. Each group contained three parallel samples. One day later, cells were ground with liquid nitrogen and peptides were processed according to the manufacturer’s protocol for the tandem mass tag (TMT) kit (Thermo Scientific, USA). The differentially expressed proteins (DEPs) were satisfied with the following conditions: unique peptides ≥1 with average ratio-fold change ≥1.5 (up-regulation) and ≤0.67 (down-regulation), as well as P values ≤ 0.05. Furthermore, the DEPs were analyzed by the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The pathway with a corrected P value < 0.05 was considered significant. As mentioned in Cell apoptosis part, insulin-like growth factor binding protein 3 (IGFBP3) (1:1000), IGFBP5 (1:1000), thrombospondin 1 (THBS1) (1:1000), apolipoprotein A1 (APOA1) (1:1000), Dickkopf 1 (DKK1) (1:1000) and Wnt5a (1:1000) which were purchased from Abcam (USA) were analyzed by WB analysis.