Journal: Advanced Science
Article Title: Lysosome‐Featured Cell Aggregate‐Released Extracellular Vesicles Regulate Iron Homeostasis and Alleviate Post‐Irradiation Endothelial Ferroptosis for Mandibular Regeneration
doi: 10.1002/advs.202505070
Figure Lengend Snippet: Lysosomal dynamics is driven by HIF‐2α signaling and regulates iron homeostasis in CAs. A) Immunostaining for HIF‐1α, HIF‐2α, or HIF‐3α (red), with nuclei stained with DAPI (blue). Scale bars, 50 µm. B–D) Quantifications of HIF‐1α, HIF‐2α, and HIF‐3α fluorescent intensity in (A). SC, unaggregated stem cell; CA, cell aggregate. E) The inhibitory efficacy of siRNAs on EPAS1 (encoding HIF‐2α) expression levels in CAs was evaluated by qRT‐PCR. F) Fluorescent staining of AIE‐lysosome (red) in CAs, with nuclei stained with Hoechst (blue). Scale bars, 50 µm. G) Quantification of AIE‐lysosome fluorescent intensity in (F). H) Fluorescent staining of the pH indicator, lysosensor DND‐189 (green) in CAs, with nuclei stained with Hoechst (blue). Scale bars, 50 µm. I) Quantification of DND‐189 fluorescent intensity in (H). siCtrl, siRNA negative control; siEPAS1, siRNA oligonucleotides of endothelial PAS domain‐containing protein 1 ( EPAS1 ). (J) Fluorescent staining of AIE‐lysosome (red), with nuclei stained with Hoechst (blue). Scale bars, 50 µm. K) Quantification of AIE‐lysosome fluorescent intensity in (J). L) Fluorescent staining of DND‐189 (green) in CAs, with nuclei stained with Hoechst (blue). Scale bars, 50 µm. M) Quantification of DND‐189 fluorescent intensity in (L). DMSO, dimethyl sulfoxide; PT2385, a HIF‐2α inhibitor. N) Fluorescent staining of the ferrous iron (Fe 2+ ) indicator, FerroOrange (red), with nuclei stained with Hoechst (blue). Scale bars, 50 µm. O) Quantification of FerroOrange fluorescent intensity in (N). P) Intracellular Fe 2+ content was analyzed by a colorimetric method. BafA1, bafilomycin A1, a lysosomal V‐ATPase inhibitor. Q) Immunostaining for Col1a1 or fibronectin (red), with nuclei stained with DAPI (blue). Scale bars, 50 µm. R,S) Quantifications of Col1a1 and fibronectin fluorescent intensity in (Q). T) ECM stiffness of CAs analyzed by the AFM nanoindentation test. U) Bright‐field images for the process of cell aggregation. Scale bars, 150 µm. V) Quantification of cell aggregation efficiency. Ferrozine, a Fe 2 ⁺ chelator. Results are expressed as mean ± SD. n = 3 samples per group for each experimental readout. p values were calculated using Student's t ‐test (B,C,D,E,G,I,K,M,R,S,T), one‐way ANOVA with Tukey's post hoc test O,P), or two‐way ANOVA (V). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.
Article Snippet: Bafilomycin A1 (BafA1; MedChemExpress, USA; Cat#HY‐100558), GW4869 (MedChemExpress, USA; Cat#HY‐19363), PT2385 (MedChemExpress, USA; Cat#HY‐12867), and ferrozine (MedChemExpress, USA; Cat#HY‐137805) were dissolved in dimethyl sulfoxide (DMSO; Beyotime, China; Cat#C2041S‐2).
Techniques: Immunostaining, Staining, Expressing, Quantitative RT-PCR, Negative Control
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