ho 1 cat p62 beclin1 lc3 ambra1 tomm20 pink1  (Proteintech)

Journal: Advanced Science

Article Title: Small Extracellular Vesicles Orchestrate Cisplatin‐Induced Ototoxicity: Potential Biomarker and Targets Discovery

doi: 10.1002/advs.202502627

Figure Lengend Snippet: ROS accumulation, apoptosis, and inflammatory responses of auditory HCs in the ex vivo cisplatin‐induced ototoxicity model. A) The flow chart of ex vivo cochlear explant culture with 50 µM cisplatin. B,C) Representative confocal images of cochlear explants treated with various durations of 50 µM cisplatin (including 24 h, 36 h, and 48 h) are presented, including the complete cochlear explants (B) and local segments of the apical, middle, and basal turns of the cochlear explants (C). The control group of cochlear explants was cultured for 48 h without drug treatment. Myosin7a (cyan) marked HCs. Scale bars are 200 µm (B) and 50 µm (C). D) A quantitative analysis of HCs in each cochlear turn at different cisplatin treatment periods is shown, expressed as the number of HCs per 100 µm of the basilar membrane. Data are shown as mean ± SD, with “*” indicating p < 0.05, “**” indicating p < 0.01, and “***” indicating p < 0.001. “n” represents the number of cochlear explants counted. E) The levels of TOMM20 protein, phosphorylated SAPK/JNK, cleaved‐Caspase3, and cleaved‐Caspase9 in the cochlear explants were all significantly increased after cisplatin‐induced injury at 48 h. β‐Actin served as the reference protein. F) The mRNA levels of Bax , Caspase3 , Caspase8 , Caspase9 , and Tomm20 were significantly increased when the cochlea was exposed to cisplatin for 48 h; n = 3. All results are presented as the mean ± SD, with statistical significance indicated as follows: “*” for p < 0.05, “**” for p < 0.01, and “***” for p < 0.001. G) Immunostaining with cleaved‐Caspase3 (red fluorescence) and anti‐Myosin7a (green fluorescence) showed that HCs in the cisplatin group exhibited a high level of the pro‐apoptosis factor. Scale bar, 20 µm. H) Mito‐Sox fluorescence staining labeled mitochondrial ROS levels in living HCs, showing significant ROS accumulation and oxidative stress in HCs after cisplatin administration. Scale bar, 20 µm.

Article Snippet: Antibodies against the ROS‐related protein TOMM20 (Proteintech, 11802‐1‐AP, 1:20 000), the inflammation‐related protein phosphorylated (p‐)SAPK/JNK (Cell Signaling Technology, #4668, 1:1000), and the pro‐apoptosis protein markers cleaved‐Caspase9 (Cell Signaling Technology, #9502, 1:1000) and cleaved‐Caspase3 (Cell Signaling Technology, #9661, 1:1000) were used for the analysis of the cochlear explants by western blotting, with β‐Actin (Abmart, P30002M, 1:10 000) serving as the internal reference.

Techniques: Ex Vivo, Control, Cell Culture, Membrane, Immunostaining, Fluorescence, Staining, Labeling