Journal: International immunopharmacology

Article Title: Oxidative stress induces ferroptosis in tendon stem cells by regulating mitophagy through cGAS-STING pathway.

doi: 10.1016/j.intimp.2024.112652

Figure Lengend Snippet: Fig. 1. Ferroptosis is involved in the oxidative damage of TSCs induced by H2O2. (A-B) Through Western blot and RT-PCR, the levels of GPX4, SLC7A11 protein and mRNA were assessed with or without the ferroptosis inhibitor Fer-1 (10 μM). (C) Detection of mitochondrial ultrastructure by TEM. (D) TSC cells were stained with Prussian blue. The levels of (E) Iron, (F) GSH, (G) MDA in TSC cells was examined using the appropriate test kits. By using ELISA assays, the level of (H) 4-HNE and (I) LPO were determined. (J) The ROS intensity in TSC cells was evaluated using ROS probe. (K) Western blot detected the protein levels of tenomodulin (n = 3); ***p < 0.001 and ###p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: To evaluate intracellular 4-HNE, the ELISA kit (CUSA BIO; # CSBEQ027232RA) was utilized.

Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Staining, Enzyme-linked Immunosorbent Assay

Journal: International immunopharmacology

Article Title: Oxidative stress induces ferroptosis in tendon stem cells by regulating mitophagy through cGAS-STING pathway.

doi: 10.1016/j.intimp.2024.112652

Figure Lengend Snippet: Fig. 3. Inhibition of mitophagy attenuates ferroptosis induced by H2O2 in TSCs. TSCs cells were pretreated with Mdivi-1 (1 μM) for 2 h and then subjected to the prescribed concentration of H2O2 (100 μM) for a period of 24 h. (A) MTT experiment were used to test cell viability. (B) Using the relevant kits, the levels of GSH in TSC cells were measured. (C–D) To measure the level of intracellular iron, Prussian blue staining and an Iron Colorimetric Assay Kit were utilized. (E) MDA levels in TSC cells were determined using the relevant kits. (F-G) To measure the amount of 4-HNE and LPO in TSC cells, ELISA assays were employed. (H) The ROS con centration in TSC cells was measured using a ROS probe. (I) Western blot detected the protein levels of Tenomodulin (n = 3); **p < 0.01, ***p < 0.001, #p < 0.05, ##p < 0.01, and ###p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: To evaluate intracellular 4-HNE, the ELISA kit (CUSA BIO; # CSBEQ027232RA) was utilized.

Techniques: Inhibition, Concentration Assay, Staining, Colorimetric Assay, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: International immunopharmacology

Article Title: Oxidative stress induces ferroptosis in tendon stem cells by regulating mitophagy through cGAS-STING pathway.

doi: 10.1016/j.intimp.2024.112652

Figure Lengend Snippet: Fig. 6. Interfering with cGAS attenuates ferroptosis induced by H2O2 in TSCs. TSC cells were treated with H2O2 for 24 h after receiving cGAS siRNA transfection for 12 h. (A) MTT experiment were used to test cell viability. (B) By using appropriate kits, the levels of GSH in TSC cells were determined. (C-D) To measure the level of intracellular iron, Prussian blue staining and an Iron Colorimetric Assay Kit were utilized. (E) To measure the amount of ROS present in TSC cells, a ROS probe was employed. (F) MDA levels in TSC cells were determined using the relevant kits. (G-H) To measure the amount of 4-HNE and LPO in TSC cells, ELISA assays were employed. (I) Western blot detected the protein levels of Tenomodulin. (n = 3); *p < 0.05, **p < 0.01, ***p < 0.001, #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: To evaluate intracellular 4-HNE, the ELISA kit (CUSA BIO; # CSBEQ027232RA) was utilized.

Techniques: Transfection, Staining, Colorimetric Assay, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Arthritis Research & Therapy

Article Title: Oxidative stress impairs energy metabolism in primary cells and synovial tissue of patients with rheumatoid arthritis

doi: 10.1186/s13075-018-1592-1

Figure Lengend Snippet: Synovial tissue (ST) angiogenesis, oxidative stress and cellular bioenergetics. To support the concept that oxidative stress, angiogenesis and energy metabolism are interconnected processes that co-exist during the inflammation milieu, double-immunofluorescence staining was performed. ST slides were co-incubated with primary mouse antibody against human 4-hydroxy-2-nonenal (4-HNE) and with primary rabbit antibodies against angiogenic factors (vascular endothelial growth factor [VEGF], angiopoietin 2 [Ang2], tyrosine kinase receptor [Tie2]), glycolytic proteins (glyceraldehyde 3-phosphate dehydrogenase [GAPDH], pyruvate kinase isozyme 2 [PKM2], glucose transporter 1 [GLUT1]) and a mitochondrial marker (adenosine triphosphate synthase subunit β [ATP5B]). Representative merged immunofluorescence images demonstrate examples of co-localisation ( yellow ) of 4-HNE with VEGF, Ang2, Tie2, GAPDH, PKM2, GLUT1 and ATP5B. Cells stained green are positive for 4-HNE only; cells stained red are positive only for VEGF, Ang2, Tie2, GAPDH, PKM2, GLUT1 and ATP5B. Arrows indicate examples of co-localisation. Magnification of photomicrographs × 20, insets show high-power magnification of co-localisation. Representative images show single immunofluorescence of 4-HNE, VEGF, Ang2, Tie2, GAPDH, PKM2, GLUT1 and ATP5B along with their controls. Isotype-matched antibodies are shown in Additional file : Figure S3 and Additional file : Figure S4

Article Snippet: ST sections were fixed with acetone for 10 minutes and co-incubated with primary mouse antibody against human 4-HNE (GENTAUR, Kampenhout, Belgium) and with primary rabbit antibodies against VEGF, Ang2, Tie2, ATP5B and glucose transporter 1 (GLUT1) (all from Abcam), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Trevigen, Gaithersburg, MD, USA) and pyruvate kinase isozyme 2 (PKM2) (Abgent, San Diego, CA, USA).

Techniques: Double Immunofluorescence Staining, Incubation, Marker, Immunofluorescence, Staining