Journal: Journal of Pharmaceutical Analysis

Article Title: Naringenin boosts Parkin-mediated mitophagy via estrogen receptor alpha to maintain mitochondrial quality control and heal diabetic foot ulcer

doi: 10.1016/j.jpha.2025.101333

Figure Lengend Snippet: Naringenin (NAR) alleviates oxidative stress and inhibition of autophagy induced by high glucose (HG). (A) HaCaT cells were treated with NAR (0.12, 0.37, 1.1, and 3.3 μM) for 24 h after being induced with HG (50 mM) for 24 h. Then, HaCaT cells were incubated with dihydroethidium (DHE) probes for 30 min, and the reactive oxygen species (ROS) levels in each group were detected using inverted fluorescence microscope. (B) HaCaT cells were incubated with DHE probes for 30 min, and the fluorescence intensity of DHE at different excitation wavelengths was subsequently detected using a multifunctional microplate detection platform. The 535 nm/370 nm ratio was quantified to assess ROS levels. (C) HaCaT cells were incubated with 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide (JC-1) dye for 30 min, and the JC-1 fluorescence was then examined using inverted fluorescence microscope. JC-1 aggregates (red) represent mitochondria with normal membrane potential, while JC-1 monomers (green) represent mitochondria with membrane potential depolarization. (D) The levels of oxidative stress-related proteins, including nicotinamide adenine dinucleotide phosphate (NAD(P)H):quinone oxidoreductase 1 (NQO1), superoxide dismutase 2 (SOD2), SOD1, and glutaredoxin 1 (GRX1), in HaCaT cells were measured by Western blotting. (E) Representative immunohistochemistry (IHC) staining images of NQO1 in wounds of mice at day 9 post-puncture. (F) The levels of sequestosome-1 (P62) and microtubule-associated proteins 1A/1B light chain 3C (LC3) in HaCaT cells were detected by Western blotting. (G) HaCaT cells were immunostained with antibody against LC3 and visualized using confocal microscope. (H) The LC3 puncta in the cells were quantified by ImageJ. Data are presented as mean ± standard deviation (SD). ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001, compared with the control group. GAPDH: glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: After blocking with 5% fat-free milk in Tris-buffered saline containing 0.1% Tween 20, the PVDF membrane was incubated with primary antibodies against β-actin (Santa Cruz Biotechnology), β-tubulin (Biodragon, Suzhou, China), p-H2A histone family member X (p-γH2AX) (Bioss, Beijing, China), adenosine triphosphate (ATP) synthase F1 subunit alpha (ATP5F1A) (Sangon Biotech Co., Ltd.), Cav-1 (Sangon Biotech Co., Ltd.), cytochrome C oxidase subunit 4 (COX IV) (Proteintech), dynamin-1-like protein (DRP1) (Sangon Biotech Co., Ltd.), ERα (Proteintech), ERβ (Sangon Biotech Co., Ltd.), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Proteintech), glutaredoxin 1 (GRX1) (Proteintech), histone H3 (Proteintech), heat shock protein 60 (HSP60) (Sangon Biotech Co., Ltd.), LaminB1 (Sangon Biotech Co., Ltd.), LC3 (MBL), mitofusin 2 (MFN2) (Proteintech), cytochrome C oxidase subunit 2 (MT-CO2) (Sangon Biotech Co., Ltd.), nicotinamide adenine dinucleotide (NADH) dehydrogenase (ubiquinone) iron-sulfur protein 4 (Ndufs4) (Sangon Biotech Co., Ltd.), nuclear factor-κB (NF-κB) (Proteintech), NQO1 (Sangon Biotech Co., Ltd.), nuclear respiratory factor 1 (NRF1) (Proteintech), cyclin-dependent kinase inhibitor 1A (P21) (Proteintech), sequestosome-1 (P62) (Proteintech), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) (Proteintech), PTEN-induced putative kinase 1 (PINK1) (Novus Biologicals, Littleton, CO, USA), p-NF-κB (S536) (Cell Signaling Technology), p-PINK1 (S228) (Thermo Fisher Scientific Inc.), succinate dehydrogenase complex flavoprotein subunit A (SDHA) (Proteintech), superoxide dismutase 1 (SOD1) (Proteintech), SOD2 (Proteintech), transcription factor A mitochondrial (TFAM) (Proteintech), translocase of inner mitochondrial membrane 23 (Tim23) (BD Biosciences, San Jose, CA, USA), Tomm20 (Proteintech), and voltage-dependent anion-selective channel protein 1 (VDAC1) (Sangon Biotech Co., Ltd.) overnight at 4 °C.

Techniques: Inhibition, Incubation, Fluorescence, Microscopy, Membrane, Western Blot, Immunohistochemistry, Standard Deviation, Control

Journal: International Journal of Molecular Sciences

Article Title: Green Synthesized Titanium Oxide Nanoparticles Promote Salt Tolerance in Soybean

doi: 10.3390/ijms26178309

Figure Lengend Snippet: Immunoblot analysis of proteins involved in soybean treated with TiO 2 NPs under salt stress. Six treatments were performed: control, CS-TiO 2 NPs, GS-TiO 2 NPs, salt, CS-TiO 2 NPs + salt, and GS-TiO 2 NPs + salt. Proteins extracted from root and hypocotyl of soybean were separated on SDS-polyacrylamide gel by electrophoresis. Proteins were transferred onto PVDF membranes. The membranes were cross-reacted with anti-superoxide dismutase, peroxiredoxin, ascorbate peroxidase, and glutathione reductase antibodies. The integrated densities of the bands were calculated using ImageJ software. Data analysis and statistical calculations are the same as in (**, p < 0.01; *, p < 0.05).

Article Snippet: After blocking, the PVDF membrane was cross-reacted with the primary antibodies for 30 min. As the primary antibodies, anti-V ATPase (Agrisera, Vännäs, Sweden), ascorbate peroxidases [ ], glutathione reductase (Agrisera), Cu/Zn superoxide dismutase (Proteintech, Rosemont, IL, USA), and peroxiredoxin [ ] antibodies were used.

Techniques: Western Blot, Control, Polyacrylamide Gel Electrophoresis, Software