Journal: Redox biology

Article Title: Dysregulation of sphingolipid and cholesterol homeostasis imposes oxidative stress in human spermatozoa.

doi: 10.1016/j.redox.2025.103669

Figure Lengend Snippet: Fig. 2. MβCD increases PI3K phosphorylation, while inhibition of S1PR or NOS impairs MβCD-induced tyrosine phosphorylation. Spermatozoa were incubated for 3.5 h at 37 ◦C under various conditions: untreated (−), treated with 10 % v/v fetal cord serum ultrafiltrate (FCSu), treated with 0.5 mM methyl- β-cyclodextrin (MβCD), and treated with MβCD in combination with (a) 40 μM VPC 23019 (S1PR1/3 inhibitor) or (b) L-NAME (Nitric Oxide Synthase inhibitor). (a) Immunoblotting analysis revealed that VPC 23019 at 40 μM decreased PI3K phosphorylation (P-PI3K) in spermatozoa incubated with 0.5 mM of MβCD. (b) Immunoblotting showed that L-NAME treatment decreased tyrosine phosphorylation (P-Tyr) in sperm samples treated with FCSu and MβCD. The signal intensity for each lane was normalized to the silver-stain optical density value for accurate quantification. α-Tubulin loading controls were performed for direct comparison with the silver stain loading controls. The data represent sperm samples from four different healthy donors (n = 4). Statistical analysis was performed using ANOVA with Tukey’s test: *p ≤0.05, **p ≤0.01, and ***p ≤0.001.

Article Snippet: Rabbit polyclonal anti-phospho-PI3K (P-PI3K) antibodies were purchased from Cell Signaling (#4228) (Beverly, MA, USA).

Techniques: Phospho-proteomics, Inhibition, Incubation, Western Blot, Silver Staining, Comparison