Journal: Science Advances

Article Title: Microphysiological model of PIK3CA-driven vascular malformations reveals a role of dysregulated Rac1 and mTORC1/2 in lesion formation

doi: 10.1126/sciadv.ade8939

Figure Lengend Snippet: ( A ) Representative images (maximum intensity projections) of PIK3CA E542K vascular networks treated with DMSO, rapamycin (1 μM) or alpelisib (2.5 μM) for 3 days. Vascular networks were labeled with fibrin (magenta), DAPI (white), and actin (cyan). End points (orange) were highlighted in 3D renderings of segmented vessels. Scale bars, 100 μm. ( B to E ) Quantification of cell number (B), ECM morphology (C), mean vessel length (D), and vessel branching (E) in GFP control, rapamycin-treated PIK3CA E542K vascular networks, or alpelisib-treated PIK3CA E542K vascular networks ( n ≥ 3; means ± SEM; one-way ANOVA with Tukey post test). ( F ) Western blot and quantification of relative p-AKT1(Thr 308 ), p-AKT1(Ser 473 ), p-S6K(Thr 389 ), and p-ERK1/2(Thr 202 /Tyr 204 ) levels in PIK3CA E542K cells treated with DMSO, rapamycin, or alpelisib for 24 hours ( n ≥ 3; mean ± SEM; one-way ANOVA with Tukey post test, * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001).

Article Snippet: Standard immunoblotting protocols were performed for Western blotting with primary antibodies used at the following concentrations: anti-p110α (1:1000; Cell Signaling Technology, #4249), anti–p-AKT-Thr 308 (1:1000, Cell Signaling Technology, #9275), anti–p-AKT-Ser 473 (1:1000; Cell Signaling Technology, #9271), anti-AKT (1:1000; Cell Signaling Technology, #9272), anti–pS6K-Thr 389 (1:1000; Cell Signaling Technology, #9205), anti-S6K (1:1000; Cell Signaling Technology, #9202), anti–pERK1/2-Thr 202 /Tyr 204 (1:1000; Cell Signaling Technology, #9101), anti-ERK1/2 (1:1000; Cell Signaling Technology), anti-Rac1/2/3 (1:1000; Cell Signaling Technology, #2465), and anti–glyceraldehyde-3-phosphate dehydrogenase (1:2000; Cell Signaling Technology, #2118).

Techniques: Labeling, Western Blot

Journal: Science Advances

Article Title: Microphysiological model of PIK3CA-driven vascular malformations reveals a role of dysregulated Rac1 and mTORC1/2 in lesion formation

doi: 10.1126/sciadv.ade8939

Figure Lengend Snippet: ( A ) Western blot analysis of relative p-AKT1(Thr 308 ), p-AKT1(Ser 473 ), p-S6K(Thr 389 ), and p-ERK1/2(Thr 202 /Tyr 204 ) levels in PIK3CA E542K cells treated with the indicated inhibitors. ( B ) Representative confocal images (maximum intensity projections) of PIK3CA E542K treated with DMSO, trametinib (MEK1/2 inhibitor, 1 μM) or torkinib (mTORC1/2 inhibitor, 5 μM) for 3 days. Scale bars, 100 μm. ( C to F ) Quantification of relative p-AKT1(Thr 308 ), p-AKT1(Ser 473 ), p-S6K(Thr 389 ), and p-ERK1/2 levels in (A) ( n ≥ 3; means ± SEM; one-way ANOVA with Tukey post test). ( G and H ) Quantification of matrix (G) and vessel branching morphology (H) in (B) ( n ≥ 3; mean ± SEM; one-way ANOVA with Tukey post test, * P ≤ 0.05; ** P ≤ 0.01; ***P ≤ 0.001; **** P ≤ 0.0001).

Article Snippet: Standard immunoblotting protocols were performed for Western blotting with primary antibodies used at the following concentrations: anti-p110α (1:1000; Cell Signaling Technology, #4249), anti–p-AKT-Thr 308 (1:1000, Cell Signaling Technology, #9275), anti–p-AKT-Ser 473 (1:1000; Cell Signaling Technology, #9271), anti-AKT (1:1000; Cell Signaling Technology, #9272), anti–pS6K-Thr 389 (1:1000; Cell Signaling Technology, #9205), anti-S6K (1:1000; Cell Signaling Technology, #9202), anti–pERK1/2-Thr 202 /Tyr 204 (1:1000; Cell Signaling Technology, #9101), anti-ERK1/2 (1:1000; Cell Signaling Technology), anti-Rac1/2/3 (1:1000; Cell Signaling Technology, #2465), and anti–glyceraldehyde-3-phosphate dehydrogenase (1:2000; Cell Signaling Technology, #2118).

Techniques: Western Blot

Journal: Cell Metabolism

Article Title: Increased fidelity of protein synthesis extends lifespan

doi: 10.1016/j.cmet.2021.08.017

Figure Lengend Snippet:

Article Snippet: The following antibodies were used at the indicated dilutions: H3 (Cell Signaling Technology; 4499S; 1:2000), pS6K (Cell Signaling Technology; 9206S; 1:1000), total S6K (kind gift from Linda Partridge laboratory; 1:1000), p4EBP (Cell Signaling Technology, 2855S; 1:500), non-phospho4E-BP (Cell Signaling Technology; 4923S; 1:500), peIF2α (Cell Signaling Technoology; 3398S; 1:1000), eIF2S1(abcam; ab26197; 1:1000); GAPDH (GeneTex; #GTX100118; 1:2000); anti-puromycin [3RH11] (Kerafast; Equation 0001; 1:2000).

Techniques: Virus, Recombinant, Protease Inhibitor, Lysis, Staining, SYBR Green Assay, Plasmid Preparation, Western Blot, Bradford Protein Assay, Luciferase, Reporter Assay, Transfection, Control, Mutagenesis, Amplification, Software