Journal: Renal Failure

Article Title: Efficacy of nicotiflorin in ameliorating septic acute kidney injury: the role of PINK1/parkin in mitochondrial restoration and oxidative stress reduction

doi: 10.1080/0886022X.2026.2624206

Figure Lengend Snippet: The effect of nicotiflorin on mitophagy in vivo. Mice were treated with/without LPS (10 mg/kg) and/or nicotiflorin (50 mg/kg) for 24 h. (A) After sacrifice of mice, kidney tissues were excised and underwent IF staining to detect PINK1 and LC3 signals (×400 magnification, scale bar: 100 µm). (B-F) Protein levels of PINK1, Parkin, LC3II/LC3I and p62 in kidney tissues were measured by Western blot. β-actin served as the loading control. *** p < 0.001 vs. Control; ^ p < 0.05, ^^ p < 0.01 vs. Nicotiflorin; # p < 0.05, ### p < 0.001 vs. LPS. n = 3, measurement data expressed as mean ± standard deviation. Difference comparisons among multiple groups were analyzed using one-way analysis of variance. PINK1: PTEN-induced putative protein kinase 1; LPS: lipopolysaccharide.

Article Snippet: Mitophagy Detection Kit (MD01, Dojindo, Tokyo, Japan) was used for detecting changes in mitophagy in NRK-52E cells as previously described [ ].

Techniques: In Vivo, Staining, Western Blot, Control, Standard Deviation

Journal: Renal Failure

Article Title: Efficacy of nicotiflorin in ameliorating septic acute kidney injury: the role of PINK1/parkin in mitochondrial restoration and oxidative stress reduction

doi: 10.1080/0886022X.2026.2624206

Figure Lengend Snippet: The effect of nicotiflorin on mitophagy in LPS-treated NRK-52E cells. NRK-52E were treated with/without LPS (10 µg/ml) and/or nicotiflorin (75 µg/ml) for 24 h. (A–B) The cells were treated with JC-10 fluorescent probe and analyzed by flow cytometer to measure mitochondrial membrane potential. (C) The mitophagy in the cells was determined by Mitophagy Dye combined with confocal microscope (×200 magnification, scale bar: 100 µm). (D–E) The degree of mitochondria fused to lysosomes was analyzed by flow cytometry after the cells were treated with Mitophagy Dye and Lyso Dye. * p < 0.05, *** p < 0.001 vs. Control; ^^ p < 0.01, ^^^ p < 0.001 vs. Nicotiflorin; # p < 0.05, ### p < 0.001 vs. LPS. n = 3, measurement data expressed as mean ± standard deviation. Difference comparisons among multiple groups were analyzed using one-way analysis of variance. LPS: lipopolysaccharide.

Article Snippet: Mitophagy Detection Kit (MD01, Dojindo, Tokyo, Japan) was used for detecting changes in mitophagy in NRK-52E cells as previously described [ ].

Techniques: Flow Cytometry, Membrane, Microscopy, Control, Standard Deviation

Journal: Renal Failure

Article Title: Efficacy of nicotiflorin in ameliorating septic acute kidney injury: the role of PINK1/parkin in mitochondrial restoration and oxidative stress reduction

doi: 10.1080/0886022X.2026.2624206

Figure Lengend Snippet: The regulatory mechanism of nicotiflorin in apoptosis, oxidative stress and mitophagy in LPS-treated NRK-52E cells. NRK-52E cells were treated with/without LPS (10 µg/ml) and/or nicotiflorin (75 µg/ml) for 24 h. (A–L) Western blot was performed to measure protein levels of Bcl-2, Bax, Cleaved caspase-3, Cyt C, Nrf2, KEAP1, PINK1, Parkin, LC3II/LC3I and p62 in the cells. β-actin served as the loading control. * p < 0.05, *** p < 0.001 vs. Control; ^^ p < 0.01, ^^^ p < 0.001 vs. Nicotiflorin; ## p < 0.01, ### p < 0.001 vs. LPS. n = 3, measurement data expressed as mean ± standard deviation. Difference comparisons among multiple groups were analyzed using one-way analysis of variance. Bcl-2: B-cell lymphoma-2; Bax: BCL2 Associated X; Cyt C: Cytochrome C; Nrf2: NF-E2-related factor-2; KEAP1: kelch-like ECH associated protein 1; PINK1: PTEN-induced putative protein kinase 1; LPS: lipopolysaccharide.

Article Snippet: Mitophagy Detection Kit (MD01, Dojindo, Tokyo, Japan) was used for detecting changes in mitophagy in NRK-52E cells as previously described [ ].

Techniques: Western Blot, Control, Standard Deviation

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:

Article Title: Skin-stage Schistosomula of Schistosoma mansoni Produce an Apoptosis-inducing Factor That Can Cause Apoptosis of T Cells *

doi: 10.1074/jbc.M201344200

Figure Lengend Snippet: Following incubation, apoptosis was measured by annexin V and propidium iodide staining and counting 500 –1000 cells under a fluorescence microscope (A), by measuring caspase-3 activity in various subset of lymphocytes using PhiPhiLux substrate and counting 500 –1000 cells under a fluorescence microscope (B), and by evaluating DNA fragmentation in different subsets after treatment with DNA digestion buffer and separation on a 1% agarose gel (C). Data presented are representative of one of three to five similar experiments using five to seven mice per group in each experiment. A and B show means ± S.D. of the percentage of positive cells. * and **, p < 0.05 and p < 0.01 compared with the medium control, respectively.

Article Snippet: Apoptotic cells around the parasites in the skin of mice were evaluated by TUNEL staining using an in situ apoptosis detection kit (R&D Systems, Minneapolis, MN) following the instructions of the manufacturer.

Techniques: Incubation, Staining, Fluorescence, Microscopy, Activity Assay, Agarose Gel Electrophoresis, Control

Journal:

Article Title: Skin-stage Schistosomula of Schistosoma mansoni Produce an Apoptosis-inducing Factor That Can Cause Apoptosis of T Cells *

doi: 10.1074/jbc.M201344200

Figure Lengend Snippet: 1 × 106 Thy1.2+ cells isolated from skin-draining lymph nodes of naive mice were cultured with 60 μg/ml ES products or ES products plus 5 μg/ml recombinant Fas-Fc fusion protein for 24 h. Apoptosis was evaluated by determining the percentage of annexin V-positive cells. *, p < 0.01 compared with the medium control.

Article Snippet: Apoptotic cells around the parasites in the skin of mice were evaluated by TUNEL staining using an in situ apoptosis detection kit (R&D Systems, Minneapolis, MN) following the instructions of the manufacturer.

Techniques: Isolation, Cell Culture, Recombinant, Control

Journal:

Article Title: Skin-stage Schistosomula of Schistosoma mansoni Produce an Apoptosis-inducing Factor That Can Cause Apoptosis of T Cells *

doi: 10.1074/jbc.M201344200

Figure Lengend Snippet: ES products were divided into three fractions, fraction 1 (Fr 1; <3 kDa), fraction 2 (Fr 2; 3–30 kDa), and fraction 3 (Fr 3; >30 kDa). Bands in fraction 2 were again separated by gel filtration. Each peak (at 60 μg/ml) was then tested for its ability to induce apoptosis of T cells (isolated from the skin-draining lymph nodes of naive mice) by staining for annexin V. Only two peaks (23 and 28 kDa) are shown. About 500 cells were counted under a fluorescence microscope. Values are representative of one of three similar experiments and are means ± S.D. of the percentage of positive cells. * and **, p < 0.05 and p < 0.001 compared with the medium control (M), respectively. ES products from normal schistosomula were used as a positive control.

Article Snippet: Apoptotic cells around the parasites in the skin of mice were evaluated by TUNEL staining using an in situ apoptosis detection kit (R&D Systems, Minneapolis, MN) following the instructions of the manufacturer.

Techniques: Filtration, Isolation, Staining, Fluorescence, Microscopy, Control, Positive Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Reduction in the developmental potential of intrathymic T cell progenitors with age.

doi: 10.4049/jimmunol.173.1.245

Figure Lengend Snippet: FIGURE 4. Proliferation and survival of ETP from young and old mice. Thymocytes were freshly isolated from young and old mice and stained for ETP in combination with anti-Ki-67 or annexin V labeling to determine their proliferation or survival status, respectively. A, ETP were labeled with anti-Ki-67 or isotype control Abs following fixation and permeabilization. The upper panel shows representative data for single young and old ani- mals. The lower panel indicates data pooled from the indicated number of different aged mice at each time point. B, The level of apoptosis in ETP was evaluated by annexin V labeling. The upper panel shows representative data for single young and old animals. The lower panel indicates data pooled from the indicated number of different aged mice at each time point. Means and SDs are shown for n 2–6 animals as indicated. , p 0.02; , p 0.002; #, p 0.005; ##, p 0.0005.

Article Snippet: The frequency of apoptotic cells in freshly harvested thymocyte populations was assessed by labeling with annexin V using the Apoptosis Detection kit as described by the manufacturer (R&D Systems, Minneapolis, MN) in combination with Abs to various cell surface determinants.

Techniques: Isolation, Staining, Labeling, Control

Journal: Research (Washington, D.C.)

Article Title: Targeting the LSD1-G9a-ER Stress Pathway as a Novel Therapeutic Strategy for Esophageal Squamous Cell Carcinoma.

doi: 10.34133/2022/9814652

Figure Lengend Snippet: Figure 4: Integrative transcriptomics analyses of ESCC cells following pharmacological or genetic inhibition of LSD1 and G9a. (a) RNA-seq analysis was performed on ESCC cells in which either LSD1 expression was knocked down using shRNA or LSD1 was inhibited with SP2509; differentially expressed genes were determined by comparing with ESCC cells expressing a control nonsilencing shRNA (shNC) and vehicle-treated ESCC cells, respectively, and are summarized using Venn diagrams. (b) Gene Set Enrichment Analysis of the overlapping differentially regulated genes in (a). (c) Heatmap of the differentially expressed genes in the “cell death,” “apoptosis,” and “ER stress” pathways. (d–f) Same as (a–c), except G9a was knocked down using shRNA or inhibited with UNC0642. (g–i) Same as (a–c), except both LSD1 and G9a were knocked down with shRNA or both LSD1 and G9a were inhibited with SP2509 and UNC0642.

Article Snippet: The following reagents were also used: hematoxylin and eosin (H&E) staining kit (Beyotime Biotech), TdT in situ apoptosis detection kit (R&D Systems, Minneapolis, MN), TRIzol reagent (Pufei Biotech, Shanghai, China), SYBR Green Supermix (Bimake, Houston, TX), RIPA buffer (Beyotime Biotech), protease inhibitors (Bimake), BCA protein assay kit (Beyotime Biotech), polyvinylidene fluoride (PVDF) membranes (Bio-Rad, Hercules, CA), Pierce ECL System (Thermo Scientific, Waltham, MA), EdU Kit (Ruibo Biotechnology Co., Ltd., Guangzhou, China), cell cycle staining kit (Multi Sciences Biotech, Hangzhou, China), annexin V-FITC/PI apoptosis detection kit (Multi Sciences Biotech), and PrimeScript RT reagent kit (Takara, Kyoto, Japan).

Techniques: Inhibition, RNA Sequencing, Expressing, shRNA, Control

Journal: Research (Washington, D.C.)

Article Title: Targeting the LSD1-G9a-ER Stress Pathway as a Novel Therapeutic Strategy for Esophageal Squamous Cell Carcinoma.

doi: 10.34133/2022/9814652

Figure Lengend Snippet: Figure 5: Inhibiting both LSD1 and G9a in ESCC cells induces S-phase arrest and apoptosis. (a) Summary of the percentage of ESCC cells in the G1, S, or G2 phase after the indicated treatments for 2 days. SP2509 5 μM, UNC0642 5 μM. (b) Western blot analysis of the indicated cell cycle-associated proteins in ESCC cells treated as indicated. (c) EdU staining of ESCC cells treated with vehicle, 3 μM SP2509, 1.2 μM UNC0642, or both for 2 days; the nuclei were counterstained with Hoechst 33342. (d) Apoptosis analysis of ESCC cells after the indicated treatments for 2 days. (e) Western blot analysis of the indicated apoptosis-associated proteins in ESCC cells treated as indicated for 2 days. (f) Quantification of the indicated proteins measured in (e), expressed relative to vehicle-treated cells. (g) Representative images of ESCC cells treated with vehicle, 10 μM SP2509, 10 μM UNC0642, or both for 2 days. (h–k) Representative transmission electron microscopy images of ESCC cells treated as in (g); mitochondria (M) and endoplasmic reticulum (ER) are indicated. The scale bars are 5 μm in (h), 2 μm in (i–k). ∗p < 0:05, ∗∗p < 0:01, and ∗∗∗p < 0:001 (unpaired Student’s t-test).

Article Snippet: The following reagents were also used: hematoxylin and eosin (H&E) staining kit (Beyotime Biotech), TdT in situ apoptosis detection kit (R&D Systems, Minneapolis, MN), TRIzol reagent (Pufei Biotech, Shanghai, China), SYBR Green Supermix (Bimake, Houston, TX), RIPA buffer (Beyotime Biotech), protease inhibitors (Bimake), BCA protein assay kit (Beyotime Biotech), polyvinylidene fluoride (PVDF) membranes (Bio-Rad, Hercules, CA), Pierce ECL System (Thermo Scientific, Waltham, MA), EdU Kit (Ruibo Biotechnology Co., Ltd., Guangzhou, China), cell cycle staining kit (Multi Sciences Biotech, Hangzhou, China), annexin V-FITC/PI apoptosis detection kit (Multi Sciences Biotech), and PrimeScript RT reagent kit (Takara, Kyoto, Japan).

Techniques: Western Blot, Staining, Transmission Assay, Electron Microscopy