s1p  (TargetMol)

Journal: The FASEB Journal

Article Title: ORMDL Proteins Turnover via Proteasome and Autophagy Is Cell‐Type Dependent and Tied to Ceramide Homeostasis

doi: 10.1096/fj.202502924RR

Figure Lengend Snippet: ORMDLs reduction does not correlate with phosphorylated sphingolipid levels and is unaffected by direct dhS1P or S1P treatment. (A) Quantification of phosphorylated sphingolipids by LC‐ESI‐MS/MS in HEK293 cells treated with low or high concentrations of myriocin, FB 1 , or their combination, (B) together with relative quantification of ORMDLs, SPTLC1, SPTLC2, and phosphorylated AKT (S473) protein levels. (C) Analysis of ORMDLs, SPTLC1, SPTLC2, and the pAKT S473 /AKT ratio in HEK293 cells following a 6‐h desensitization period and subsequent 30‐ or 60‐min stimulation with dhS1P or S1P. In both (A) and (B), protein levels were normalized to total protein loading based on Ponceau S staining. (D) Representative time‐lapse images of HEK293 cells showing morphological responses to dhS1P or S1P stimulation. Scale bar: 100 μm. Data in (A‐C) are presented as geometric means ± GSEM ( N = 3). Statistical significance was assessed by one‐way ANOVA followed by Tukey's post hoc test. Significance levels: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****); n.s., not significant.

Article Snippet: Briefly, 50 μL of homogenate was transferred to a 1.5 mL microcentrifuge tube (Cat# 780420, Brand GmbH, Wertheim, Germany), mixed with 25 μL of MilliQ water, and loaded with internal standards: 50 ng dhS1P d17:0 (Cat# 860655), 50 ng S1P d17:1 (Cat# 860641), and 25 ng C1P d18:1/C12:0 (Cat# 860531), each from Avanti Polar Lipids, dissolved in 5 μL of chloroform/methanol solution (2:1, v/v).

Techniques: Tandem Mass Spectroscopy, Quantitative Proteomics, Staining

Journal: The FASEB Journal

Article Title: ORMDL Proteins Turnover via Proteasome and Autophagy Is Cell‐Type Dependent and Tied to Ceramide Homeostasis

doi: 10.1096/fj.202502924RR

Figure Lengend Snippet: ORMDLs reduction does not correlate with phosphorylated sphingolipid levels and is unaffected by direct dhS1P or S1P treatment. (A) Quantification of phosphorylated sphingolipids by LC‐ESI‐MS/MS in HEK293 cells treated with low or high concentrations of myriocin, FB 1 , or their combination, (B) together with relative quantification of ORMDLs, SPTLC1, SPTLC2, and phosphorylated AKT (S473) protein levels. (C) Analysis of ORMDLs, SPTLC1, SPTLC2, and the pAKT S473 /AKT ratio in HEK293 cells following a 6‐h desensitization period and subsequent 30‐ or 60‐min stimulation with dhS1P or S1P. In both (A) and (B), protein levels were normalized to total protein loading based on Ponceau S staining. (D) Representative time‐lapse images of HEK293 cells showing morphological responses to dhS1P or S1P stimulation. Scale bar: 100 μm. Data in (A‐C) are presented as geometric means ± GSEM ( N = 3). Statistical significance was assessed by one‐way ANOVA followed by Tukey's post hoc test. Significance levels: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****); n.s., not significant.

Article Snippet: Sphingolipid concentrations of S1P, dhC1P, and C1P were determined by single‐point calibration using external standards [ ]: 50 ng S1P d18:1 (Cat# 860492), 50 ng dhC1P d18:0/C16:0 (Cat# 860522), and 50 ng C1P d18:1/C16:0 (Cat# 860533), all from Avanti Polar Lipids.

Techniques: Tandem Mass Spectroscopy, Quantitative Proteomics, Staining

Journal: The FASEB Journal

Article Title: ORMDL Proteins Turnover via Proteasome and Autophagy Is Cell‐Type Dependent and Tied to Ceramide Homeostasis

doi: 10.1096/fj.202502924RR

Figure Lengend Snippet: Regulation of ORMDLs stability in human RPE‐1 cells and mouse BMMCs. (A) Levels of ORMDLs, SPTLC1, SPTLC2, and total d18:1 ceramides (measured by LC‐ESI‐MS/MS) were assessed in human RPE‐1 cells and mouse BMMCs after 24‐h treatment with 10 μM myriocin or 10 μM FB 1 , and compared with untreated controls. (B) Quantification of ORMDLs, SPTLC1, SPTLC2, and the LC3‐II/LC3‐I ratio was performed in untreated RPE‐1 cells and BMMCs, and compared with cells treated for 24 h with 50 μM CQ or 10 μM MG132, as determined by immunoblotting. (C) Effects of p97/VCP inhibition by CB‐5083 on ORMDLs, SPTLC1, SPTLC2, the LC3‐II/LC3‐I ratio, ATF4, and p62 levels were assessed in RPE‐1 cells and BMMCs. Protein levels in (A‐C) were normalized to total protein loading using Ponceau S staining. Data are presented as geometric mean ± GSEM ( N = 4). Statistical analysis was performed using one‐way ANOVA with Tukey's post hoc test. Significance levels: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****); n.s., not significant.

Article Snippet: Sphingolipid concentrations of S1P, dhC1P, and C1P were determined by single‐point calibration using external standards [ ]: 50 ng S1P d18:1 (Cat# 860492), 50 ng dhC1P d18:0/C16:0 (Cat# 860522), and 50 ng C1P d18:1/C16:0 (Cat# 860533), all from Avanti Polar Lipids.

Techniques: Tandem Mass Spectroscopy, Western Blot, Inhibition, Staining