Journal: Materials Today Bio

Article Title: Extracellular biogenic nanoscale mitochondria reprogram the wound microenvironment via ROS scavenging independent of cellular uptake

doi: 10.1016/j.mtbio.2026.103023

Figure Lengend Snippet: MSC-mt internalization promotes mitophagy activation under oxidative stress (A-B) Flow cytometric analysis of mitophagy in L929 cells co-cultured with fluorescently labeled MSC-mt under H 2 O 2 -induced oxidative stress. Mitophagy levels are shown for total cells as well as stratified mt transfer + and mt transfer − subpopulations, showing preferential mitophagy activation in mt transfer + cells. (C-D) Western blot analysis of mitophagy- and survival-related signaling proteins in flow-sorted mt transfer + and mt transfer − L929 cells following co-culture with fluorescently labeled MSC-mt under oxidative stress. Blots show phosphorylated PINK1 (S228), total PINK1, Parkin, total p62, phosphorylated p62 (S349 and S403), pAKT, OXPHOS components, and TOM20, highlighting enhanced PINK1–Parkin signaling and mitophagy-associated p62 processing in mt transfer + cells. (E) Flow cytometric assessment of mitophagy in total, mt transfer + , and mt transfer − populations following co-culture with PINK1-deficient MSC-derived mitochondria (siPINK1-mt) under oxidative stress, showing attenuated mitophagy activation compared with control MSC-mt. (F) Representative immunofluorescence images of L929 cells under control, H 2 O 2 , and H 2 O 2 + MSC-mt conditions, showing depolarized mitochondria (mitoPeDPP, green) and mitophagy signals (mitophagy, red), indicating increased mitophagic engagement under oxidative stress with MSC-mt transfer. Scale bar = 20 μm. (G–J) Flow cytometric analysis of depolarized mitochondria (mitoPeDPP) and mitophagy in L929 cells under H 2 O 2 stimulation with or without fluorescently labeled MSC-mt co-culture. (G) Representative flow cytometry plots. (H) Quantification of the proportions of mitoPeDPP + , mitophagy + , and double-positive cell populations. (I) Mean fluorescence intensity (MFI) of mitophagy signals, with stratification by mt transfer + and mt transfer − populations. (J) MFI of mitoPeDPP signals, with stratification by mt transfer + and mt transfer − populations. All experiments were independently repeated three times (n = 3) and representative images are shown. Data are presented as mean ± SEM. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ns, not significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: To investigate the role of PINK1 in MSC-mt–mediated mitophagy, small interfering RNA (siRNA) targeting murine PINK1 (siPINK1) was synthesized by Shanghai Generay Co., Ltd. For knockdown in MSCs, cells at ∼60% confluence were transfected with siPINK1 (final concentration: 50 nM) using LipofectamineTM RNAiMAX Transfection Reagent (Thermo Fisher, Cat# 13778030) according to the manufacturer's protocol.

Techniques: Activation Assay, Cell Culture, Labeling, Western Blot, Co-Culture Assay, Derivative Assay, Control, Immunofluorescence, Flow Cytometry, Fluorescence