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 mitigate oxidative damage and improve mitochondrial function in H 2 O 2 -treated skin fibroblasts. (A–B) Representative flow cytometry plots and quantification of Annexin V/PI staining for apoptosis after H 2 O 2 stimulation, showing that MSC-mt significantly reduce fibroblast apoptosis, whereas rotenone-pretreated mitochondria (mt(R)) exhibit markedly attenuated protective effects. (C–D) Representative plots and quantification of mitoSOX staining for mitochondrial ROS after 180 min H 2 O 2 exposure, showing that MSC-mt suppress mitochondrial ROS accumulation, an effect largely lost following rotenone pretreatment. (E–F) Representative plots and quantification of TMRE staining for mitochondrial membrane potential (ΔΨm) after 180 min H 2 O 2 treatment, showing preservation of mitochondrial membrane potential by MSC-mt but not by mt(R). (G–H) Representative plots and quantification of SA-β-gal staining for cellular senescence after 180 min H 2 O 2 treatment, showing reduced senescence burden in MSC-mt–treated cells, with diminished efficacy observed in the mt(R) group. (I) Representative images and quantification of colony formation assays on day 8 post-treatment, indicating improved long-term proliferative capacity following MSC-mt treatment, an effect substantially weakened following rotenone pretreatment. (J) Heatmap of differentially expressed genes identified by a wound healing PCR array following 180 min of H 2 O 2 stimulation, showing that MSC-mt partially reverse H 2 O 2 -induced transcriptional alterations associated with stress response and repair pathways, an effect attenuated when using mt(R). (K) Representative images of mouse apoptosis protein array analysis after 180 min of H 2 O 2 treatment, showing that MSC-mt partially restore the expression of key anti-apoptotic and pro-survival signaling proteins suppressed by oxidative stress, including phosphorylated AKT (Ser473), BAD (Ser112), ERK (T202), and IκBα (S32), with reduced restoration observed in the mt(R) group. (L) Western blot analysis of phosphorylated AKT at Ser473 expression after 180 min of H 2 O 2 exposure, showing restoration of pro-survival signaling by MSC-mt, which is attenuated in the mt(R) group. All experiments were independently repeated three times (n = 3). Data are presented as mean ± SEM. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ns, not significant.

Article Snippet: After blocking with 5% non-fat milk, membranes were incubated overnight at 4 °C with the following primary antibodies: anti-PINK1 (CST, Cat# 6946), anti-NIX (CST, Cat# 12396), anti-TOM20 (CST, Cat# 42406), anti-USP30 (Proteintech, Cat# 15402-1-AP), Total OXPHOS Rodent WB Antibody Cocktail (Abcam, Cat# ab110413), anti-phospho-AKT (Ser473) (CST, Cat# 4060), and anti-β-actin (CST, Cat# 4967).

Techniques: Flow Cytometry, Staining, Membrane, Preserving, Protein Array, Expressing, Western Blot

Journal: The Journal of Experimental Medicine

Article Title: MYCT1–IFITM2/3 interaction links endothelial endolysosomal trafficking to white adipose tissue expansion

doi: 10.1084/jem.20251497

Figure Lengend Snippet: MYCT1 limits endothelial mTORC1 signaling, related to Figs. 3 and 4. (A) Flow cytometry gating strategy (CD45 neg CD31 + ) for sorting of ECs from mesenteric fat for scRNA-seq. (B) Dot plot of markers for the indicated clusters. Color code: scaled average expression level in each cluster; the dot size denotes the percent of cells in each cluster expressing the given gene. (C) Number of differentially expressed genes (DEGs) between wild-type and Myct1 ecKO cell clusters. (D) Volcano plot of DEGs between the wild-type and Myct1 ecKO mice in the BEC cluster. Mat2a gene was selected for scRNA-seq validation. Mat2a, methionine adenosyltransferase 2A. (E) MYCT1 protein levels in human primary ECs. Western blot analysis for the indicated proteins. HPMECs, human pulmonary ECs; HUVECs, human umbilical vein ECs; HIECs, human intestinal ECs. (F and G) MYCT1 antibody and siRNAs validation for identification of endogenous human MYCT1 protein. Human primary ECs were transfected with two different MYCT1 targeting siRNAs. (F) Staining of ECs for MYCT1 (black) and DAPI (blue). Arrow, not transfected EC. Scale bar, 50 μm. (G) Western blot analysis showing MYCT1 migration profile and siRNA specificity. (H) MYCT1 knockdown increases phosphorylation of S6 but does not affect AKT and ERK1/2 phosphorylation status. Western blot analysis for the indicated proteins. (I) Quantification of p-S6 Ser240/244 levels normalized to total S6 (tot-S6). P = 0.037 (*). (J) Quantification of p-AKT Ser473 levels normalized to total AKT (tot-AKT). P > 0.05. (K) Quantification of p-AKT Thr308 levels normalized to total AKT (tot-AKT). P > 0.05. (L) Quantification of p-ERK1/2 Thr202/Tyr204 levels normalized to total ERK1/2 (tot-ERK1/2). P > 0.05. (M) Quantification of MYCT1 levels normalized to vinculin. P = 0.001 (*). (I–M) n = 5 independent experiments; paired t tests. (N) MYCT1 knockdown increases phosphorylation of p70/S6 kinase (p70/S6K), a key downstream effector of mTORC1 signaling, in response to amino acids. Western blot for the indicated proteins. (O) Quantification of data shown in N. n = 2 independent experiments; mean ± SD; two-way ANOVA with Tukey’s multiple comparison test, P = 0.0194 (*). (P) MYCT1 knockdown hyperactivates mTORC1 signaling in response to amino acids. 2 days after siRNA transfection, confluent ECs were serum- and growth factor–starved overnight, then starved in PBS for 1 h before 30-min stimulation with amino acids, glucose, growth factors, FBS, their combination, or PBS as control. Staining of ECs for p-S6 (gray), VE-cadherin (magenta), and DAPI (blue). Scale bar, 50 μm. Source data are available for this figure: .

Article Snippet: Rabbit monoclonal anti-phospho-Akt (Ser473) , Cell Signaling Technology , Cat# 4060, RRID:AB_2315049.

Techniques: Flow Cytometry, Expressing, Biomarker Discovery, Western Blot, Transfection, Staining, Migration, Knockdown, Phospho-proteomics, Comparison, Control