Journal: Journal of Orthopaedic Translation

Article Title: Targeting ANT1 to regulate PINK1/Parkin-mediated mitophagy is an effective treatment of trauma-induced tendon heterotopic ossification

doi: 10.1016/j.jot.2025.08.002

Figure Lengend Snippet: InTPCs have higher levels of PINK1/Parkin-mediated mitophagy and osteogenic differentiation than TSPCs . (A) Representative TEM images showing healthy mitochondria (white arrow) and mitophagy (red arrow) in the TSPC and inTPC groups when undifferentiated and when exposed to ODM for two days and two weeks, respectively. Scale bar = 1 μm (original magnification) and 200 nm (insert magnification of the boxed area). (B) Quantification of the number of mitochondria per TEM field. (C) Quantification of the number of APs and ALs per TEM field. (D) Representative JC-1 fluorescence images of TSPCs and inTPCs after two days of osteogenic induction (red: 561 nm/green: 488 nm). Scale bar = 100 μm. (E–G) Quantification of the JC-1 fluorescence intensity ratio. (H) Representative colocalization images of mitochondrial and lysosomes in TSPCs and inTPCs after two days of osteogenic induction (red: 594 nm/green: 488 nm). Scale bar = 10 μm (original magnification) and 2 μm (insert magnification of the boxed area). (I, J) Pixel matching colocalization analysis in TSPCs (I) and inTPCs (J). (K) Pearson's correlation and Overlap coefficient of colocalization. (L, M) qRT–PCR analysis of the mitophagy-related genes Pink1 , Prkn , LC3b , and p62 (L) and osteogenic-related genes Ocn , Runx2 , and Alpl (M) in TSPCs and inTPCs after two weeks of osteogenic induction. (N, O) Western blotting analysis (N) and quantification (O) of mitophagy-related proteins, PINK1, Parkin, LC3BⅡ, and p62 in TSPCs and inTPCs after two weeks of osteogenic induction. (P, Q) Western blotting analysis (P) and quantification (Q) of osteogenic-related proteins, OCN, RUNX2, and ALP in TSPCs and inTPCs after two weeks of osteogenic induction. (R) ALP staining of TSPCs and inTPCs under weeklong osteogenic induction. Scale bar = 200 μm. (S) Quantification of ALP staining. (T) ARS staining of TSPCs and inTPCs under two weeks of osteogenic induction. Scale bar = 200 μm. (U) Quantification of ARS staining. ∗ p < 0.05, ns, not significant.

Article Snippet: Mitochondria in cell samples were stained using the MitoMP Detection Kit (JC-1) (Dojindo, MT09, Japan) at 37 °C for 30 min.

Techniques: Fluorescence, Quantitative RT-PCR, Western Blot, Staining

Journal: Journal of Orthopaedic Translation

Article Title: Targeting ANT1 to regulate PINK1/Parkin-mediated mitophagy is an effective treatment of trauma-induced tendon heterotopic ossification

doi: 10.1016/j.jot.2025.08.002

Figure Lengend Snippet: Elamipretide inhibits PINK1/Parkin-mediated mitophagy and osteogenic differentiation of inTPCs . (A) Representative TEM images showing healthy mitochondria (white arrow) and mitophagy (red arrow) in inTPCs treated with vehicle or 1 μM elamipretide when exposed to ODM for two days and two weeks, respectively. Scale bar = 1 μm (original magnification) and 200 nm (insert magnification of the boxed area). (B) Quantification of the number of mitochondria per TEM field. (C) Quantification of the number of APs and ALs per TEM field. (D) Representative JC-1 fluorescence images of inTPCs treated with vehicle or 1 μM elamipretide after two days of osteogenic induction (red: 561 nm/green: 488 nm). Scale bar = 100 μm. (E–G) Quantification of the JC-1 fluorescence intensity ratio. (H) Representative colocalization images of mitochondria and lysosomes in inTPCs treated with vehicle or 1 μM elamipretide after two days of osteogenic induction (red: 594 nm/green: 488 nm). Scale bar = 10 μm (original magnification) and 2 μm (insert magnification of the boxed area). (I, J) Pixel matching colocalization analysis in inTPCs treated with vehicle (I) or 1 μM elamipretide (J). (K) Pearson's correlation and Overlap coefficient of colocalization. (L, M) qRT–PCR analysis of the mitophagy-related genes Pink1 , Prkn , LC3b , and p62 (L) and the osteogenic-related genes Ocn , Runx2 , and Alpl (M) in inTPCs treated with vehicle or 1 μM elamipretide after two weeks of osteogenic induction. (N, O) Western blotting analysis (N) and quantification (O) of mitophagy-related proteins PINK1, Parkin, LC3BⅡ, and p62 in inTPCs treated with vehicle or 1 μM elamipretide after two weeks of osteogenic induction. (P, Q) Western blotting analysis (P) and quantification (Q) of osteogenic-related proteins OCN, RUNX2, and ALP in inTPCs treated with vehicle or 1 μM elamipretide after two weeks of osteogenic induction. (R) ALP staining of inTPCs treated with vehicle or 1 μM elamipretide under weeklong osteogenic induction. Scale bar = 200 μm. (S) Quantification of ALP staining. (T) ARS staining of inTPCs treated with vehicle or 1 μM elamipretide under two weeks of osteogenic induction. Scale bar = 200 μm. (U) Quantification of ARS staining. ∗ p < 0.05, ns, not significant.

Article Snippet: Mitochondria in cell samples were stained using the MitoMP Detection Kit (JC-1) (Dojindo, MT09, Japan) at 37 °C for 30 min.

Techniques: Fluorescence, Quantitative RT-PCR, Western Blot, Staining

Journal: Ecotoxicology and environmental safety

Article Title: Mechanistic insights into severe pulmonary inflammation caused by silica stimulation: The role of macrophage pyroptosis.

doi: 10.1016/j.ecoenv.2023.114975

Figure Lengend Snippet: Fig. 2. Silica exposure induces mitochondrial depolarization and disrupts energy homeostasis in RAW-ASC cells. RAW-ASC cells were primed with LPS for 6 h then treated with nano-silica, micro-silica, and ATP for 4 h, respectively. (A) Representative images of JC-1 staining showing the alteration of mitochondrial membrane potential between groups. Red, JC-1 aggregates; Green, JC-1 monomers. Scale bar = 50 µm. (B) Quantitative analysis and comparison of fluorescence intensity in A between groups (n = 6). (C) Measurement and comparison of intracellular ROS content between groups (n = 6). (D) Measurement and comparison of intracellular ATP content between groups (n = 4). ns, not significant, *P < 0.05, **P < 0.01.

Article Snippet: The mouse macrophage cell line RAW264.7 cells stably expressing ASC (RAW-ASC) were obtained from Invivogen (San Diego, CA, USA); Dulbecco’s modified Eagle’s medium of high glucose (DMEM), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody, and goat antirabbit IgG-HRP conjugated secondary antibody were obtained from Servicebio (Wuhan, China); Silica nanoparticles, penicillinstreptomycin, mitochondrial Membrane Potential Assay Kit with JC-1, and adenosine triphosphate (ATP) were purchased from Solarbio (Beijing, China); Brilliant blue G (BBG), lipopolysaccharides (LPS), reactive oxygen assay kit, calcium content chromogenic assay kit, ATP assay kit, and Calcein/PI cell assay kit were purchased from Beyotime (Shanghai, China); KCl was obtained from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China); Fetal bovine serum (FBS) was obtained from BI (Israel); Silica particles of micro-size, and fast green FCF were obtained from Sigma-Aldrich (St. Louis, MO, USA); Primary antibodies of NLRP3, P2X7, Pannexin-1, and IL-1β were purchased from ABclonal (Wuhan, China).

Techniques: Staining, Membrane, Comparison, Fluorescence

Journal: Nanomaterials

Article Title: Water Filtration Membranes Based on Non-Woven Cellulose Fabrics: Effect of Nanopolysaccharide Coatings on Selective Particle Rejection, Antifouling, and Antibacterial Properties

doi: 10.3390/nano11071752

Figure Lengend Snippet: ( A ) Confocal microscopy micrographs showing BSA adsorption on the surface of the unmodified ( a ), CNC impregnated ( b ), T-CNF impregnated ( c ) and ChNC impregnated ( d ) nonwoven fabrics stained with the Qubit Protein Assay Kit after their incubation with BSA solution for 6 h at pH 7. ( B ) Confocal microscopy micrographs showing E. coli colonization and viability on the surface of the unmodified ( e ), CNC impregnated ( f ), T-CNF impregnated ( g ) and ChNC impregnated ( h ) nonwoven fabrics stained with Live/Dead BacLight Bacterial Viability Kit. Live cells were green stained by SYTO 9 and dead cells were red-stained by PI.

Article Snippet: The proteins were visualized using a Qubit Protein Assay Kit and with a Zeiss LSM 780 Confocal fluorescence microscope (Carl Zeiss MicroImaging GmbH, Oberkochen, Germany).

Techniques: Confocal Microscopy, Adsorption, Staining, Qubit Protein Assay, Incubation

Journal: Frontiers in Immunology

Article Title: Pharmacological Activation Of Aldehyde Dehydrogenase 2 Protects Against Heatstroke-Induced Acute Lung Injury by Modulating Oxidative Stress and Endothelial Dysfunction

doi: 10.3389/fimmu.2021.740562

Figure Lengend Snippet: Silencing ALDH2 augmented heat stress-induced activation of NF-κB, ROS production and apoptosis in HUVECs in vitro. HUVECs were transfected with ALDH2 siRNA or control siRNA (scramble) before heat stress induction (42°C for 2 h). (A) ALDH2 protein expression was measured by immunoblotting ( n = 5). (B) The ALDH2 activity in cell lysate was determined by measuring NADH production based on the O.D. absorbance at 450 nm in a microplate reader ( n = 5). (C) Measurement of ROS production based on DCF fluorescence using a fluorescence microplate reader with an excitation wavelength of 488 nm and an emission wavelength of 535 nm ( n = 5). (D) Measurement of cellular ROS production based on DHE fluorescence using a fluorescence microplate reader with an excitation wavelength of 518 nm and an emission wavelength of 606 nm ( n = 5). (E) The viability of HUVECs was measured by the MTT assay based on the O.D. absorbance at 570 nm in a microplate reader ( n = 5). (F) The levels of apoptosis were measured by the TUNEL assay as determined fluorescence microscopy. The percentage of apoptotic cells was determined based on the number of TUNEL-positive cells among the total number of cells ( n = 5). (G) The levels of senescence were measured by β-galactosidase activity detection using bright field microscopy ( n = 5). (H) Detection of mitochondrial dysfunction by the JC-1 assay, revealing a decrease in the mitochondrial membrane potential (ΔΨm) in live cells as determined by fluorescence microscopy and fluorescence microplate reader. The ΔΨm level is expressed as the merge of the red and green channels, and the data were quantified as the ratio of red fluorescence intensity to the green fluorescence intensity ( n = 5). (I) The protein and 4-HNE levels in lung homogenates were measured by immunoblotting. Densitometric analysis was conducted with imaging processing software. The data were quantified by normalization to GAPDH; phosphorylated proteins were normalized to total proteins ( n = 5). The data are expressed as the mean ± SD. Statistical significance is indicated as * p < 0.05.

Article Snippet: To assess mitochondrial function in HUVECs after HS, JC-1 (5,5’,6,6’-tetrachloro-1,1’,3,3’- tetraethylbenzimidazolcarbocyanine iodide) staining (BD Biosciences, 551302) was performed and assessed by fluorescence microscopy (Nikon Eclipse 50i) at a magnification of 200×.

Techniques: Activation Assay, In Vitro, Transfection, Expressing, Western Blot, Activity Assay, Fluorescence, MTT Assay, TUNEL Assay, Microscopy, Imaging, Software

Journal: Frontiers in Immunology

Article Title: Pharmacological Activation Of Aldehyde Dehydrogenase 2 Protects Against Heatstroke-Induced Acute Lung Injury by Modulating Oxidative Stress and Endothelial Dysfunction

doi: 10.3389/fimmu.2021.740562

Figure Lengend Snippet: Effects of Alda-1 on HS-induced vascular inflammation, ROS and apoptosis in vitro . Alda-1 (20 μM) was added to HUVECs for 6 h before HS (42°C for 2 h). (A) The ALDH2 activity in cell lysate was determined by measuring NADH production based on the O.D. absorbance at 450 nm in a microplate reader ( n = 5). (B) Measurement of ROS production based on DCF fluorescence as determined by using a fluorescence microplate reader with an excitation wavelength of 488 nm and an emission wavelength of 535 nm ( n = 5). (C) Measurement of cellular ROS production based on DHE fluorescence as determined using a fluorescence microplate reader with an excitation wavelength of 518 nm and an emission wavelength of 606 nm ( n = 5). (D) The viability of HUVECs was measured by the MTT assay based on the O.D. absorbance at 570 nm in a microplate reader ( n = 5). (E) The levels of apoptosis were measured by the TUNEL assay as determined by fluorescence microscopy. The percentage of apoptotic cells was determined based on the number of TUNEL-positive cells among the total number of cells. (F) The levels of senescence were measured by β-galactosidase activity detection using bright field microscopy ( n = 5). (G) Detection of mitochondrial dysfunction by the JC-1 assay, revealing that the mitochondrial membrane potential (ΔΨm) was decreased in live cells as determined by fluorescence microscopy and a fluorescence microplate reader. The ΔΨm level is expressed as the merge of the red and green channels, and the data were quantified as the ratio of red fluorescence intensity to green fluorescence intensity ( n = 5). (H) The protein and 4-HNE levels in lung homogenates were measured by immunoblotting. Densitometric analysis was conducted with imaging processing software. The data were quantified by normalization to GAPDH; phosphorylated proteins were normalized to total proteins ( n = 5). The data are expressed as the mean ± SD. Statistical significance is indicated as * p < 0.05.

Article Snippet: To assess mitochondrial function in HUVECs after HS, JC-1 (5,5’,6,6’-tetrachloro-1,1’,3,3’- tetraethylbenzimidazolcarbocyanine iodide) staining (BD Biosciences, 551302) was performed and assessed by fluorescence microscopy (Nikon Eclipse 50i) at a magnification of 200×.

Techniques: In Vitro, Activity Assay, Fluorescence, MTT Assay, TUNEL Assay, Microscopy, Western Blot, Imaging, Software