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: Advanced Science

Article Title: Microglial Fkbp5 Impairs Post‐Stroke Vascular Integrity and Regeneration by Promoting Yap1‐Mediated Glycolysis and Oxidative Phosphorylation

doi: 10.1002/advs.202512499

Figure Lengend Snippet: Fkbp5 knockdown inhibits the function and metabolic status of stroke‐VAM after OGD/R in vitro. A,B) Representative immunoblots images (A) and quantitative data (B) of M2 markers (CD206, IL‐10, and IL‐4) of BV2 cells receiving NT siRNA ( n = 6) or si‐Fkbp5 siRNA ( n = 6) under OGD/R condition. C,D) Flow plot (C) and mean fluorescence intensity (mFI) (D) of CD206 in BV2 cells receiving NT siRNA ( n = 7) or si‐Fkbp5 siRNA ( n = 7) before OGD/R treatment. E,F) Flow plot (E) and mean fluorescence intensity (mFI) (F) of IgG‐latex in BV2 cells after administration of NT siRNA ( n = 5) or si‐Fkbp5 siRNA ( n = 5) after OGD/R condition. G,H) Representative pictures (G) and quantitative data of uptake of IgG‐latex in BV2 cells after administration of NT siRNA ( n = 6) or si‐Fkbp5 siRNA ( n = 6) after OGD/R condition. I–L) Representative images (I) and morphological quantitative analysis (including mean perimeter (J), mean form factor (K), and mean branch length (L)) of primary microglial cells after NT siRNA or Fkbp5 knockdown treatment followed by OGD/R. n = 8 for each group. M,N) Representative JC‐1 flow plot (M) and relative ratio (N) of JC‐1 aggregates to JC‐1 monomers of control BV2 cells ( n = 6) and Fkbp5‐downregulated BV2 cells ( n = 6) after OGD/R injury. O,P) Representative micrograph (O) and quantitative data (P) of JC‐1 monomers and JC‐1 aggregates of BV2 cells receiving NT siRNA ( n = 8) or si‐Fkbp5 siRNA ( n = 8) under OGD/R condition. Q,R) Representative micrograph (Q) and quantitative data (R) of mitoSOX of BV2 cells receiving NT siRNA ( n = 8) or si‐Fkbp5 siRNA ( n = 8) under OGD/R condition. S) Lactate concentration in extracellular medium released from BV2 cells after administration of NT siRNA ( n = 6) or si‐Fkbp5 siRNA ( n = 6) under OGD/R condition. T,U) Representative immunoblots (T) and quantitative data (U) of glycolysis enzymes expression in BV2 cells after administration of NT siRNA ( n = 7) or si‐Fkbp5 siRNA ( n = 7) under OGD/R condition. Data are presented as mean ± SD. unpaired t ‐test; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.OGD/R, Oxygen Glucose Deprivation/Reperfusion; DAPI, 4′,6‐diamidino‐2‐phenylindole.

Article Snippet: Enhanced mitochondrial membrane potential assay kit with JC‐1 (C2003S, Beyotime, China) was used to measure mitochondrial membrane potential according to the user's guidelines.

Techniques: Knockdown, In Vitro, Western Blot, Fluorescence, Control, Concentration Assay, Expressing

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