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: Journal of Clinical and Translational Hepatology

Article Title: USP10 Alleviates Palmitic Acid-induced Steatosis through Autophagy in HepG2 Cells

doi: 10.14218/jcth.2022.00060

Figure Lengend Snippet: Fig. 7. USP10 activated TSC2 and JNK1 signaling pathways in HepG2 cells. HepG2 cells overexpressing USP10 were treated with PA (312.5 µM) for 24 h. Protein expression was assayed by western blotting. (A) Protein expression of tuberous sclerosis complex-2 (TSC2), mammalian target of rapamycin (mTOR), p-mTOR, ribosomal protein S6 kinase (S6K), and p-S6K. Data are means±SD, n=3 (**p<0.01). (B) Expression of C-jun N-terminal protein kinase-1 (JNK1), VPS34, ATG14, Bcl2, and p-Bcl2. Data are means±SD, n=3 (*p<0.05; **p<0.01; NS, not significant). ATG, autophagy target gene; JNK, C-jun N-terminal protein kinase; mTOR, mamma- lian target of rapamycin; S6K, ribosomal protein S6 kinase; TSC, tuberous sclerosis complex; USP, ubiquitin-specific peptidase; VPS34, phosphatidylinositol 3-kinase.

Article Snippet: Antibodies to ATG5 (cat. no. A0203), Beclin1 (cat. no. A11761), tuberous sclerosis complex (TSC)2 (cat. no. A19540), C-jun N-terminal protein kinase-1 (JNK1) (cat. no. A0288), phosphatidylinositol 3-kinase (VPS34) (cat. no. A12295), B-cell lymphoma 2 (Bcl2) (cat. no. A19693), p-Bcl2 at Ser70 (cat. no. AP0575), ribosomal protein S6 kinase (S6K) (cat. no. A4898), p-S6K at Thr389 (cat. no. AP1059) and p62 (cat. no. A7758) were provided by ABclonal (Woburn, MA, USA).

Techniques: Protein-Protein interactions, Expressing, Western Blot, Ubiquitin Proteomics