Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Everolimus Triggers Cytokine Release by Macrophages

doi: 10.1161/atvbaha.112.245381

Figure Lengend Snippet: Figure 1. mTOR inhibition affects de novo protein synthesis. A and B, Mouse macrophages were incubated in serum-containing RPMI medium (control), Earle Balanced Salt Solution (EBSS, starvation) or RPMI supplemented with 10 mmol/L everoli mus. Phospho-p70 S6 kinase (P-p70S6K, Thr389) (A), and phospho-eukaryotic initiation factor 2 (P-eIF2, Ser51) (B) were analyzed by Western blotting. **P0.01, ***P0.001 versus con trol (0 minutes) (1-way ANOVA, followed by Dunnett test, n3). C, Mouse macrophages were incubated in serum-containing RPMI (control), EBSS (starvation), or RPMI supplemented with 10 mmol/L everolimus or 10 mg/mL cycloheximide (CHX) for 2 hours in the presence of L-35S-methionine/cysteine. 35S-labeled proteins were measured with scintillation counting. *P0.05, ***P0.001 versus control (1-way ANOVA, followed by Dunnett test, n7).

Article Snippet: Macrophages were then starved in Earle Balanced Salt Solution (EBSS; Invitrogen, Carlsbad, CA) or treated with 10 mol/L everolimus (Novartis, Basel, Switzerland) in RPMI 1640 medium (Invitrogen).

Techniques: Inhibition, Incubation, Control, Western Blot, Labeling

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Everolimus Triggers Cytokine Release by Macrophages

doi: 10.1161/atvbaha.112.245381

Figure Lengend Snippet: Figure 2. Everolimus-induced mTOR inhibition stimulates phosphorylation of p38 MAPK and its substrates ATF2 and cPLA2. Mouse macrophages were starved in Earle Balanced Salt Solution (EBSS) or treated with 10 mmol/L everoli mus in RPMI medium for up to 8 hours. Phosphorylation of p38 MAPK (Thr180/ Tyr182) (A), activating transcription factor 2 (ATF2, Thr71) (B), cellular phospho lipase A2 (cPLA2) (C), and NF-B p65 (Ser536) (D) was analyzed by Western blotting. Treatment of cells with 1 mg/ mL LPS was used as positive control for NF-B p65 activation. *P0.05, ***P0.001 versus control (0 minutes) (1-way ANOVA, followed by Dunnett test, n53).

Article Snippet: Macrophages were then starved in Earle Balanced Salt Solution (EBSS; Invitrogen, Carlsbad, CA) or treated with 10 mol/L everolimus (Novartis, Basel, Switzerland) in RPMI 1640 medium (Invitrogen).

Techniques: Inhibition, Phospho-proteomics, Western Blot, Positive Control, Activation Assay, Control