Journal: Materials Today Bio

Article Title: ATG7-driven mitophagy in BMSC@CS hydrogel reprograms metabolism to boost bone regeneration

doi: 10.1016/j.mtbio.2025.102483

Figure Lengend Snippet: The effects of ATG7 overexpression in MSCs on mitochondrial function and energy metabolism. (A) Schematic illustration of the effects of ATG7 overexpression on mitochondrial function and energy metabolism in MSCs. (B) The mRNA levels of GLUT-3, GLUT-4, Ndufs1, Ndufs5, Ndufs6, Cyc1, CYTB and Uqcrfs1 were detected by RT‒qPCR in MSCs overexpressing ATG7. (C) ROS staining and (D) ROS FACs analysis of MSCs overexpressing ATG7. Scale bar = 75 μm. (E) TMRM staining and (F) TMRM FACs analysis of MSCs overexpressing ATG7. Scale bar = 75 μm. (G) lactate levels in the cell culture supernatant and (H) intracellular ATP levels were measured in MSCs with overexpression of ATG7.

Article Snippet: Intracellular ROS levels were assessed by incubating cells with 5 μM H 2 DCFDA (MCE, HY-D0940) in PBS for 30 min at 37 °C in the dark, followed by mounting with DAPI-containing medium (Solarbio, S2110) or staining with 1 mM mitochondrial tracker (MCE, HY-D1783) for 30 min ΔΨm was evaluated using 10 μM TMRM (MCE, HY-D0984A) under the same conditions.

Techniques: Over Expression, Staining, Cell Culture

Journal: Materials Today Bio

Article Title: ATG7-driven mitophagy in BMSC@CS hydrogel reprograms metabolism to boost bone regeneration

doi: 10.1016/j.mtbio.2025.102483

Figure Lengend Snippet: ATG7 Regulates Osteogenic Function via Mitochondrial Autophagy Activation in MSCs. (A) Western blot analysis of P62, Pink1, Parkin, and LC3 Ⅱ/Ⅰ protein expression in MSCs overexpressing Atg7. (B) Microscopy images and quantification of showing autophagy in BMSCs after overexpression ATG7. Mitochondria are stained green with Mitotracker, and lysosomes are stained red with Lysotracker. Yellow arrows indicate regions of mitochondrial and lysosomal co-localization. Scale bars: 25 μm. (C) Western blot analysis and quantification of P62, Pink1, Parkin, and LC3 Ⅱ/Ⅰ protein expression in Mdivi-1 (100 μM, 48 h) pretreated MSCs overexpressing ATG7. (D) RT‒qPCR analysis of osteogenic marker genes (ALP, OPN, OCN, and DMP-1) in ATG7-overexpressing MSCs pretreated with Mdivi-1 (100 μM) for 48 h. (E) ALP staining and quantitative analysis of osteoblast colony formation in Mdivi-1-pretreated MSCs overexpressing ATG7. Scale bar = 250 μm. (F) Western blot analysis and (G) quantification of AKT, p-AKT, PI3K, and p-PI3K protein expression in Mdivi-1 pretreated MSCs overexpressing ATG7. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Intracellular ROS levels were assessed by incubating cells with 5 μM H 2 DCFDA (MCE, HY-D0940) in PBS for 30 min at 37 °C in the dark, followed by mounting with DAPI-containing medium (Solarbio, S2110) or staining with 1 mM mitochondrial tracker (MCE, HY-D1783) for 30 min ΔΨm was evaluated using 10 μM TMRM (MCE, HY-D0984A) under the same conditions.

Techniques: Activation Assay, Western Blot, Expressing, Microscopy, Over Expression, Staining, Marker

Journal: Neuro-oncology

Article Title: Inhibition of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme of the nicotinamide adenine dinucleotide (NAD) salvage pathway, to target glioma heterogeneity through mitochondrial oxidative stress.

doi: 10.1093/neuonc/noab175

Figure Lengend Snippet: Fig. 4 The effects of NAMPT inhibition on mitochondrial function. (A) Western blot analysis of SirT1 expression in the GSC262, GSC811, and GSC5-22 cell lines. (B) Assessment of the oxygen consumption rate (OCR). An Agilent Seahorse assay was used to determine the effect of KPT- 9274 on the basal respiration, maximum respiration, and spare respiratory capacity of the GSC262, GSC811, and GSC5-22 cell lines and the effect of adding 500 µM NMN, a pathway intermediate for the NAD salvage pathway, to reverse the effect of KPT-9274. (C) Immunofluorescence assays for mitochondrial morphologies, numbers, and membrane potentials in GSC262, GSC811, and GSC5-22 cells exposed to KPT-9274 or the vehicle and probed with 4′,6-diamidino-2-phenylindole (DAPI), Tom20-Alexa488, and MitoTracker Red CMXRos. Scale bar = 15 µm (D) Flow cytometric assessments of the mitochondrial potential of the GSC262, GSC811, and GSC5-22 cell lines exposed to 0.1 or 1.0 µM KPT-9274 or the vehicle and measured by staining with 2 µM JC-1 dye.

Article Snippet: After overnight incubation, cells were exposed to vehicle or 0.5 μM of KPT-9274 for 72 h. After incubation with MitoTracker Red CMXRos (20 nM) (Cat# 9082; Cell Signaling Technologies) for 30 min, cells were fixed with 4% formalin, permeabilized with 1% Triton X, and stained with anti-Tom20 (Cat# 42406; Cell Signaling Technologies) followed by Alexa Fluor D ow nloaded from https://academ ic.oup.com /neuro-oncology/article/24/2/229/6321319 by guest on 27 April 2024 233Sharma et al. NAMPT inhibition and mechanisms in gliomas N eu ro- O n colog y 488-conjugated anti-rabbit F(ab’)2 fragment (Cat# 4412; Cell Signaling Technologies).

Techniques: Inhibition, Western Blot, Expressing, Immunofluorescence, Membrane, Staining

Journal: Aging Cell

Article Title: Mitochondrial S‐adenosylmethionine deficiency induces mitochondrial unfolded protein response and extends lifespan in Caenorhabditis elegans

doi: 10.1111/acel.14103

Figure Lengend Snippet: Inactivating TRMT‐10C.2 impairs protein translation and respiration in mitochondria. (a) Representative confocal microscopy images of the co‐localization of TRMT‐10C.2::GFP and MitoTracker Red in L4 transgenic worms carrying trmt‐10C . 2 :: GFP . Scale bar = 10 μm. (b) Representative immunoblots for CTC‐1 and NUO‐2 in wild‐type worms fed with empty vector (EV) and trmt‐10C . 2 RNAi bacteria (left). Densitometric quantification of CTC‐1 or NUO‐2: β‐actin ratio relative to EV groups (right). Means ± SEMs for four biological replicates. *** p < 0.001, Student's t ‐test. (c) Puromycin incorporation followed by western blot analysis using antibodies detecting puromycin, NUO‐2, and actin in the whole worm lysate and mitochondrial lysate of day 1 wild‐type worms treated with empty vector or trmt‐10C . 2 RNAi. Densitometric quantification of puromycin incorporation in the whole worm lysate and mitochondrial lysate (right). Means ± SEMs for three biological replicates. *** p < 0.001, Student's t ‐test. (d) Oxygen Consumption Rate (OCR) of L4 wild‐type worms treated with empty vector or tmt‐10C . 2 RNAi was measured by Seahorse Analysis. Data are representative of n = 3 biological replicates with 30 worms per treatment.

Article Snippet: For Mitotracker Red staining, L4 worms carrying TRMT‐10C.2::GFP were cultured on UV‐killed bacteria with 2 μM Mitotracker (Cell Signaling Technology, 8778) for 24 h. Worms were subsequently transferred to stain‐free plates for 30 min to eliminate the excess stain in the gut lumen before proceeding with image analysis.

Techniques: Confocal Microscopy, Transgenic Assay, Western Blot, Plasmid Preparation, Bacteria

Journal: Respiratory Research

Article Title: Effects of the common polymorphism in the human aldehyde dehydrogenase 2 ( ALDH2 ) gene on the lung

doi: 10.1186/s12931-017-0554-5

Figure Lengend Snippet: Functional and quantitative assessment of mitochondria in mouse tracheal and lung epithelium. MTECs and lung cells were collected from WT, Aldh2*2 Tg, and Aldh2 −/− mice, n = 4-5 mice/genotype. Cells were immunostained for the mitochondrial membrane protein, Tim23. No difference in fluorescence intensity was observed between WT ( a ) and Aldh2*2 Tg ( b ), but MTECs from Aldh2 −/− mice showed significantly lower fluorescence intensity ( c ). Quantification of fluorescence intensity from multiple cells from each genotype is shown in ( d ). Cells were also stained for MitoTracker (MT) probes and examined by fluorescence microscopy and flow cytometry. In comparison to WT cells ( e ), lung cells from Aldh2*2 Tg mice ( f ) had a higher fluorescence intensity, while the Aldh2 −/− mice ( g ) showed lower fluorescence intensity. Quantification of fluorescence intensity from multiple cells from each genotype is shown in ( h ). Not Aldh2*2 Tg ( i ) but Aldh2 −/− ( j ) showed a higher level of mitochondrial ROS than WT mice did. MTECs from WT and Aldh2 −/− mice were examined using a flux analyzer to measure and compare OCR ( k ) and ECAR ( l ) as indicators of the cellular mitochondrial respiration and glycolysis. No significant difference was observed between WT and Aldh2 −/− cells. a - c , e - g scale bars: 10 μm, nuclei were stained with DAPI ( blue )

Article Snippet: MitoTracker® Red FM and/or MitoTracker® Red CMXRos (Promega) were used according to the manufacturer’s protocol.

Techniques: Functional Assay, Membrane, Fluorescence, Staining, Microscopy, Flow Cytometry, Comparison