Journal: Antioxidants

Article Title: Pulsatilla Saponin B4 Alleviates H 2 O 2 -Induced Oxidative Stress and Apoptosis via the AMPK/Nrf2 Pathway in Bovine Mammary Epithelial Cell Models

doi: 10.3390/antiox15030294

Figure Lengend Snippet: Effects of PSB4 on T-AOC, SOD, GSH-Px, MDA, and ROS levels in H 2 O 2 -induced BMECs. BMECs were exposed to H 2 O 2 (500 μM) for 12 h, followed by incubation with PSB4 (25, 50, or 100 μg/mL) for a further 12 h. ( A – D ) The activities of T-AOC, SOD, and GSH-Px, along with MDA content, were measured in BMECs ( n = 3) ( E ) Representative confocal images of ROS staining in BMECs. Green: DCFH-DA fluorescent probe (ROS); Gray: bright-field (cell morphology); Merged: overlay of green fluorescence and bright-field. Scale bar = 20 μm. ( F ) Statistical comparison of relative fluorescence intensity among treatment groups ( n = 3). Data are presented as the mean ± standard error of the mean (SEM). Statistical significance is indicated in the figures as * p < 0.05 and ** p < 0.01.

Article Snippet: Mitochondrial membrane potential was detected by flow cytometry using a JC-1 fluorescent probe kit (Beyotime, Shanghai, China), and changes in membrane potential were evaluated based on the ratio of JC-1 aggregates to monomers.

Techniques: Incubation, Staining, Fluorescence, Comparison

Journal: Antioxidants

Article Title: Pulsatilla Saponin B4 Alleviates H 2 O 2 -Induced Oxidative Stress and Apoptosis via the AMPK/Nrf2 Pathway in Bovine Mammary Epithelial Cell Models

doi: 10.3390/antiox15030294

Figure Lengend Snippet: PSB4 demonstrated antioxidative properties by means of AMPK signaling in H 2 O 2 -treated BMECs. BMECs were first stimulated after exposure to H 2 O 2 (500 μM) for 12 h, followed by a 12 h incubation in combination with PSB4 (100 μg/mL) and CC (10 μM) in the culture medium. ( A ) Representative immunofluorescence images of Nrf2 nuclear translocation in BMECs under different treatment conditions. Red: Nrf2 (Alexa Fluor 594); blue: DAPI (nuclei). Scale bar = 10 µm; n = 3. ( B ) Representative confocal images of ROS staining in BMECs. Green: DCFH-DA fluorescent probe (ROS); Gray: bright-field (cell morphology); Merged: overlay of green fluorescence and bright-field. Scale bar = 20 μm. ( C ) Quantitative analysis of relative fluorescence intensity in BMECs across treatment groups ( n = 3). ( D ) Western blot assay of Nrf2, p-AMPK and AMPK in BMECs. The predicted molecular weights for Nrf2, AMPK, p-AMPK, and β-actin were 97 kDa, 62 kDa, 62 kDa, and 42 kDa, respectively. ( E , F ) Evaluation of the relative intensity of the respective protein bands, standardized to β-actin; quantification of the bands from the Western blot ( n = 3). ( G – J ) Gene expression changes in GCLM , HO-1 , NQO1 and GPX1 were analyzed using qRT-PCR ( n = 3). Values are expressed as the mean ± SEM. Statistical significance is indicated as follows: ** p < 0.01.

Article Snippet: Mitochondrial membrane potential was detected by flow cytometry using a JC-1 fluorescent probe kit (Beyotime, Shanghai, China), and changes in membrane potential were evaluated based on the ratio of JC-1 aggregates to monomers.

Techniques: Incubation, Immunofluorescence, Translocation Assay, Staining, Fluorescence, Western Blot, Gene Expression, Quantitative RT-PCR

Journal: Viruses

Article Title: Infectious Spleen and Kidney Necrosis Virus Triggers Ferroptosis in CPB Cells to Enhance Virus Replication.

doi: 10.3390/v17050713

Figure Lengend Snippet: Figure 3. ISKNV infection induces ferroptosis in CPB cells. (A) Transmission electron microscopy of CPB cells treated with DMSO (72 h), erastin (10 µmol/L, 72 h), and ISKNV (100 MOI, 24 h, 48 h, 72 h). Scale bars = 1 µm. (B) Analysis of Fe2+ levels in CPB cells after treatment with ISKNV (100 MOI) using the fluorescent probe FerroOrange by laser scanning confocal microscopy. Scale bars = 20 µm. (C) Quantitative analysis of the mean fluorescence intensity of (B) using Image J. (D) Analysis of intracellular ROS levels using DCFH-DA staining, and laser scanning confocal microscopy of CPB cells treated with ISKNV (100 MOI) for 24–72 h. Scale bars = 10 µm. (E) Quantitative analysis of the mean fluorescence intensity of (D) using Image J. (F–H) Detection of Fe2+, ROS, and MDA levels in cell lysates treated with ISKNV (100 MOI) for 24–72 h by microplate reader. * p < 0.05, ** p < 0.01, and *** p < 0.001, with p > 0.05 considered not significant (ns).

Article Snippet: The Fe2+ content of the cells was detected by laser scanning confocal microscopy and a microplate reader using the fluorescent probe FerroOrange (Elabscience, E-BC-F101, Wuhan, China).

Techniques: Infection, Transmission Assay, Electron Microscopy, Confocal Microscopy, Fluorescence, Staining