nitrocellulose membrane  (Bio-Rad)

Journal: Journal of Sport and Health Science

Article Title: Influence of diet-induced obesity and voluntary exercise training on cardiac lipids and mitochondrial function in mice

doi: 10.1016/j.jshs.2025.101095

Figure Lengend Snippet: Voluntary exercise alters left ventricle gene and protein expression related to mitochondrial biogenesis and dynamics in high fat- and chow-fed mice. (A) Western blot analysis to assess purity of crude mitochondria isolation where left ventricle mitochondrial pellet expresses mitochondrial marker TOM70 and COXIV and supernatant expresses calnexin. Protein expression of (B) LCLAT1 and (C) MFN2 in left ventricle-isolated crude mitochondria and (D) LCLAT1, (E) PGC-1α, and (F) MFN2 in left ventricle tissue from male VET or sedentary mice fed an HFD or chow diet. Left ventricle mRNA expression of (G) OPA1 and (H) DRP1 in male VET or sedentary mice fed an HFD or chow diet. Values were calculated relative to 18S housekeeper. Calnexin and β-actin were used as internal controls of protein loading in left ventricle samples and COXIV as internal control of protein loading in crude mitochondria samples. Analysis was performed using two-way analysis of variance with Tukey’s post hoc test for multiple comparisons. Data are expressed as mean ± standard error of the mean. (B–C) n : 5–6 per group. (D–F) n = 7 per group. (G and H) n = 9 per group. CE = chow exercise; CS = chow sedentary; COXIV = cytochrome c oxidase subunit 4; DRP1 = dynamin-related protein 1; HE = high fat diet exercise; HFD = high fat diet; HS = high fat diet sedentary; LCLAT1 = lysocardiolipin acyltransferase 1; MFN2 = mitofusin-2; OPA1 = optic atrophy 1; PGC-1α = peroxisome proliferator-activated receptor gamma coactivator-1α; TOM70 = translocase of outer mitochondria membrane 70; VET = voluntary exercise training.

Article Snippet: Immunoblotting was performed with antibodies against lysocardiolipin acyltransferase 1(LCLAT1, PA5-25627; Thermo Fisher Scientific), PGC-1α (ab191838; Abcam, Cambridge, UK), MFN2 (PA5-118059; Thermo Fisher Scientific), ANP (sc-18811; Santa Cruz, Dallas, TX, USA), tumor necrosis factor alpha (TNFα, 3707; Cell Signaling, Danvers, MA, USA), phosphorylated adenosine monophosphate-activated protein kinase (AMPK, 2531; Cell Signaling), AMPKα (2532; Cell Signaling), Perilipin 5 (PA1-46215; Thermo Fisher Scientific), translocase of outer mitochondria membrane 70 (TOM70, 65675; Cell Signaling), cytochrome c oxidase subunit 4 (COXIV, 4844; Cell Signaling), and calnexin (208880; Merck, Rahway, NJ, USA).

Techniques: Expressing, Western Blot, Isolation, Marker, Control, Membrane

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: The mitochondrial membrane potential (ΔΨm) of isolated MSC-mt was assessed using a TMRE Mitochondrial Membrane Potential Assay Kit (Beyotime, Cat# C2001S).

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