p90rsk agonist ceramide c6  (MedChemExpress)

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: MβCD promotes nitric oxide (NO) and superoxide (O 2 •- ) production in human spermatozoa. Spermatozoa were incubated for 3.5 h at 37 °C under various conditions: with (+) or without (−) 10 %v/v Fetal cord serum ultrafiltrate (FCSu) and 0.5 mM methyl β-cyclodextrin (MβCD) 40 μM VPC 23019 (S1PR1/3 inhibitor) and 0.5 mg/mL superoxide dismutase (SOD1) to assess various parameters. Intracellular NO levels were measured using DAF-2DA fluorescence, while sperm viability was evaluated using Sytox Blue fluorescence. Tyrosine phosphorylation (P-Tyr) was assessed by immunoblotting, and extracellular superoxide anion (O 2 •- ) production was quantified using MCLA chemiluminescence. ( a ) Scatter plot depicting viable spermatozoa (Sytox Blue − ) categorized based on nitric oxide (NO) production, as determined by DAF-2DA fluorescence. Spermatozoa were classified into DAF-2DA + (blue, Q4), indicating NO production and DAF-2DA − (yellow, Q3) for non–NO–producing cells. ( b ) Immunoblot analysis of P-Tyr in sperm samples incubated with sphingosine (Sph), ceramide (Cer), and 0.5 mg/mL SOD1. Spermatozoa were treated in BWW medium with or without 10 % v/v FCSu (-Δ-), with or without 0.5 mM MβCD (-◊-), and with or without 0.5 mg/mL SOD1. Chemiluminescence was measured following the addition of 20 μM MCLA, and the data show SOD-inhibitory chemiluminescence relative intensity, standardized to non-treated controls. α-Tubulin loading controls were performed for direct comparison with the silver stain loading controls. Data represent sperm samples from 4 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: Mouse monoclonal anti-P-Tyrosine (P-Tyr), clone 4G10 (#05–321 ) , nitrocellulose blotting membrane (GE10600004), Nω-Nitro- l -arginine methyl ester hydrochloride ( l -NAME) (N5751), progesterone (P0130), Pisum sativum lectin conjugated with FITC (PSA-FITC) (L0770), Superoxide Dismutase (SOD) from bovine liver (S8160), and bovine serum albumin (BSA) (A9418), C6 ceramide (d18:1/6:0) (860506), sphingosine (d18:1) (860490), and cholesterol sulfate (Chol-SO 4 ) (700016) were purchased from Millipore Sigma Canada (Oakville, ON, Canada).

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

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: High levels of cholesterol, Sphingosine, and Ceramide impair capacitation. Spermatozoa were incubated for 3.5 h at 37 °C, with (+) or without (−) ( a ) 0.5 mM methyl-β-cyclodextrin (MβCD) and increasing concentrations of cholesterol-sulfate (Chol-SO 4 ), ( b ) 10–40 μM sphingosine (Sph), or ( c ) 40–80 μM ceramide (Cer), to assess tyrosine phosphorylation (P-Tyr) fluorescence. Immunoblot analysis revealed a dose-dependent decrease in P-Tyr in human spermatozoa treated with increasing concentrations of Chol-SO 4 , Sph, and Cer compared to controls (MβCD alone, Sph 10 μM, and Cer 40 μM, respectively). All lines correspond to the same blot for sphingosine or ceramide treatments. Signal quantification was performed by normalizing each lane to its silver-stain optical density. Data represent sperm samples from 4 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: Mouse monoclonal anti-P-Tyrosine (P-Tyr), clone 4G10 (#05–321 ) , nitrocellulose blotting membrane (GE10600004), Nω-Nitro- l -arginine methyl ester hydrochloride ( l -NAME) (N5751), progesterone (P0130), Pisum sativum lectin conjugated with FITC (PSA-FITC) (L0770), Superoxide Dismutase (SOD) from bovine liver (S8160), and bovine serum albumin (BSA) (A9418), C6 ceramide (d18:1/6:0) (860506), sphingosine (d18:1) (860490), and cholesterol sulfate (Chol-SO 4 ) (700016) were purchased from Millipore Sigma Canada (Oakville, ON, Canada).

Techniques: Incubation, Phospho-proteomics, Fluorescence, Western Blot, Silver Staining

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: High levels of Sphingosine and Ceramide invoke oxidative stress and damage . Spermatozoa were incubated for 3.5 h at 37 °C with or without (Non-Treated) treatment with fetal cord serum ultrafiltrate (FCSu 10 % v/v), bovine serum albumin/bicarbonate (BSA/HCO 3 ), 10 μM and 40 μM sphingosine (Sph), or 40 μM and 80 μM ceramide (Cer). After washing, sperm samples were incubated with the following probes: ( a ) anti-8-OHdG FITC-conjugated antibody (1:1000) for DNA oxidation and Sytox Blue (0.2 μM) for viability; ( b ) MitoSOX (2 μM) for mitochondrial superoxide (O 2 •- ) production and Sytox Blue (0.2 μM); ( c ) JC-1 (2 μM) for mitochondrial membrane potential and Sytox Blue (0.2 μM); ( d ) BODIPY-C11 (5 μM) for lipid peroxidation and Sytox Blue (0.2 μM); and ( e ) immunoblotting with anti-3-nitrotyrosine (1:1000) for protein nitration. Flow cytometry analysis was performed to identify live (Q3) and dead (Q2) cells. Data are expressed as either the ratio of red/green fluorescence intensity in ( c ), indicating depolarization when the ratio decreases, or as the percentage of cells positive for 8-OHdG ( a ), O 2 •− production ( b ), and lipid peroxidation ( d ). Immunoblot analysis in ( e ) reflects the relative intensity of 3-nitrotyrosine, with each lane normalized to its silver-stain optical density for signal quantification. Results represent sperm samples from 4 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: Mouse monoclonal anti-P-Tyrosine (P-Tyr), clone 4G10 (#05–321 ) , nitrocellulose blotting membrane (GE10600004), Nω-Nitro- l -arginine methyl ester hydrochloride ( l -NAME) (N5751), progesterone (P0130), Pisum sativum lectin conjugated with FITC (PSA-FITC) (L0770), Superoxide Dismutase (SOD) from bovine liver (S8160), and bovine serum albumin (BSA) (A9418), C6 ceramide (d18:1/6:0) (860506), sphingosine (d18:1) (860490), and cholesterol sulfate (Chol-SO 4 ) (700016) were purchased from Millipore Sigma Canada (Oakville, ON, Canada).

Techniques: Incubation, Membrane, Western Blot, Nitration, Flow Cytometry, Fluorescence, Silver Staining

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: Pre-treatment of sperm with high levels of sphingolipid and cholesterol impairs FCSu-induced capacitation. Spermatozoa were first pre-treated with 40 μM sphingosine (Sph), 80 μM of Ceramide (Cer), or 1 mM cholesterol-sulfate (Chol-SO 4 ) for 15 min at 37 °C. Then the samples were washed and incubated for 3.5 h at 37 °C, without any treatment (Non-Treated) or 10 %v/v Fetal cord serum ultrafiltrate (FCSu). Immunoblot analysis reveals a decrease in tyrosine phosphorylation (P-Tyr) in sperm samples pre-treated with high levels of sphingolipids or cholesterol. Each lane was normalized to its silver-stain optical density value for accurate signal quantification. Data represent sperm samples from 4 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: Mouse monoclonal anti-P-Tyrosine (P-Tyr), clone 4G10 (#05–321 ) , nitrocellulose blotting membrane (GE10600004), Nω-Nitro- l -arginine methyl ester hydrochloride ( l -NAME) (N5751), progesterone (P0130), Pisum sativum lectin conjugated with FITC (PSA-FITC) (L0770), Superoxide Dismutase (SOD) from bovine liver (S8160), and bovine serum albumin (BSA) (A9418), C6 ceramide (d18:1/6:0) (860506), sphingosine (d18:1) (860490), and cholesterol sulfate (Chol-SO 4 ) (700016) were purchased from Millipore Sigma Canada (Oakville, ON, Canada).

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

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: Dysregulation of sphingolipid rheostat and cholesterol homeostasis promotes oxidative stress in human spermatozoa, impacting male fertility. Elevated levels of Sphingosine (Sph), Ceramide (Cer), and cholesterol-sulfate (Chol-SO 4 ) result in mitochondrial dysregulation and leakage, causing an uptick in superoxide production. The formation of hydrogen peroxide (H 2 O 2 ) and peroxynitrite (ONOO − ) can promote oxidative stress and lipid peroxidation. The release of cytotoxic byproduct 4-HNE can further exacerbate the loss of mitochondrial function, impair motility, cause DNA damage (DNA oxidation and mutations), and impair sperm capacitation. Moreover, an increase in ROS generation can directly increase DNA oxidation, impairing sperm function. More specifically, a high level of ONOO − can lead to the tyrosine-nitration of protein, which is important for sperm motility and capacitation. Altogether, dysregulated sphingolipid and cholesterol homeostasis can result in male infertility. This figure was generated using BioRender.

Article Snippet: Mouse monoclonal anti-P-Tyrosine (P-Tyr), clone 4G10 (#05–321 ) , nitrocellulose blotting membrane (GE10600004), Nω-Nitro- l -arginine methyl ester hydrochloride ( l -NAME) (N5751), progesterone (P0130), Pisum sativum lectin conjugated with FITC (PSA-FITC) (L0770), Superoxide Dismutase (SOD) from bovine liver (S8160), and bovine serum albumin (BSA) (A9418), C6 ceramide (d18:1/6:0) (860506), sphingosine (d18:1) (860490), and cholesterol sulfate (Chol-SO 4 ) (700016) were purchased from Millipore Sigma Canada (Oakville, ON, Canada).

Techniques: Nitration, Generated

Journal: bioRxiv

Article Title: Hormetic elevation of taurine restrains inflammaging by deactivating the NLRP3 inflammasome

doi: 10.1101/2025.05.27.656381

Figure Lengend Snippet: (a) Volcano plots of the 290 metabolites identified between the LPS plus ATP challenge group and control group in the bone marrow derived macrophages (BMDMs). Each dot represents a metabolite identified by LC-MS/MS. The volcano plot shows the fold-change (x-axis) versus the significance (y-axis) of the identified 290 metabolites. The significance (non-adjusted p-value) and the fold change are converted to -Log 10 (p-value) and log 2 (fold-change). The horizontal dotted lines show the cut-off of fold-change= ±1.0, and of p-value=0.05. 26 significantly regulated metabolites in response to LPS plus ATP treatment compared with baseline were heighted (up-regulated in red, down-regulated in blue (n=3, P<0.05). The right panel is the top 26 discriminating parameters in descending order of importance from the metabolomics data of BMDMs with LPS, ATP treatment and control group. The colored legend on the right indicates the relative abundance of variables, with red and blue indicating high and low values, respectively, while beige illustrates neutral values. (b) Changes in the levels of taurine and L-cystine in response to LPS and ATP stimulation in BMDMs, measured by LC-MS/MS. (c) Taurine efflux was determined in LPS-primed-BMDMs treated with 10 mM ATP, Hypotonic solution (90 mOsmolarity), 10 μM Nigericin for 45 mins, Ceramide 6, MSU for 6 hours and Imiquimod for 2 hours. (d) Intracellular and culture medium taurine levels in BMDMs primed with LPS and stimulated with ATP. (e) Western blots of cell lysates and supernatants from BMDMs stimulated with LPS and ATP and treated with taurine. These results are representative of five independent experiments. (f) ELISA analysis of IL-1β production from BMDMs stimulated with LPS (1 μg/ml, 4 hours), followed by ATP (5 mM, 45 min) treatment with or without taurine, MCC950 or KCl. (g) Intracellular taurine level following inflammasome activation in BMDMs supplemented with 100 mM taurine, 10 μM MCC950 or 45 mM KCl. (Representative of three experiments). (h-i) Production of IL-18 and LDH from BMDM stimulated with LPS and ATP and treated with taurine as measured by ELISA (h) and LDH assay (i). (j) Intracellular taurine concentration in LPS-primed BMDMs stimulated by hypotonicity in the presence of sodium chloride. (Representative of three experiments). (k) Western blots of cell lysates and supernatants from BMDMs activated with hypotonic solution in the presence of taurine or NaCl. (Representative of three experiments). (l) Production of IL-1β from BMDMs stimulated with LPS and hypotonic solution and treated with taurine. (m) Intracellular taurine level of LPS-primed BMDMs from control and Nlrp3 deficient mice following ATP or hypotonic treatment supplemented with 10 μM MCC950. (Representative of three experiments). (n) Intracellular taurine concentration in thioglycolate elicited macrophages sorted from peritoneal cavity of mice treated with LPS and ATP. These data are representative of three independent experiments. Error bars represent the mean ± SEM.

Article Snippet: Primed cells were then treated with 5 mM ATP (Sigma) for 45 min, 250 μg/ml MSU (Invitrogen) for 6 hours or 100μM Ceramide C6 (Cayman) for 6 hours, Nigericin (10 μM, Invivogen, 1h) or Imiquimod (50 μM, Invitrogen, 2h) to activate the NLRP3 inflammasome.

Techniques: Control, Derivative Assay, Liquid Chromatography with Mass Spectroscopy, Western Blot, Enzyme-linked Immunosorbent Assay, Activation Assay, Lactate Dehydrogenase Assay, Concentration Assay