Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Proteomics reveals Rictor as a noncanonical TGF-β signaling target during aneurysm progression in Marfan mice

doi: 10.1152/ajpheart.00089.2018

Figure Lengend Snippet: Identification of β3-integrin (ITβ3) as a potential mediator of rapamycin-independent component of mammalian target of rapamycin (Rictor) activation in aged Marfan syndrome (MFS) mice. A: protein-level data for α5-integrin (ITα5), β1-integrin (ITβ1), and αv-integrin (ITαv) identified from the original proteomic analysis along with a targeted reanalysis of data-independent acquisition mass spectrometry files to identify and quantify ITβ3. *mapDIA false discovery rate (FDR) of <1% from the original proteomic analysis; #independent-samples two-tailed t-test, P < 0.05 for Skyline quantification. B: Western blot confirmation of ITβ1 [n = 4, wild-type (WT) and MFS] and ITβ3 (n = 3, WT and MFS) expression in the aorta of aged mice, with integrin signals normalized to β-tubulin to account for loading differences. #Independent-samples two-tailed t-test, P < 0.05; n.s., not significant. C: quantification of fibronectin and vitronectin expression from original proteomics analysis. Statistics were performed in mapDIA software. *FDR < 1%. D: immunofluorescence analysis of vitronectin and fibronectin expression and localization in ×20 magnification images of the aorta of aged WT (n = 3) and MFS (n = 4) mice [blue, DAPI cell nuclei; red, vitronectin (Vn); green, fibronectin (Fn)]. Quantification of mean intensity for each marker is shown at the right. Independent-samples t-test P values are given on each comparison panel.

Article Snippet: Cells were plated on immobilized vitronectin (2 µg/ml, incubated 1 h and then removed, Promega, Madison, WI)-coated six-well plates at a density of 10 6 cells/well in complete DMEM (5% FBS).

Techniques: Activation Assay, Mass Spectrometry, Two Tailed Test, Western Blot, Expressing, Software, Immunofluorescence, Marker

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Proteomics reveals Rictor as a noncanonical TGF-β signaling target during aneurysm progression in Marfan mice

doi: 10.1152/ajpheart.00089.2018

Figure Lengend Snippet: Roles of vitronectin and β3-integrin (ITβ3) in mediating transforming growth factor (TGF)-β-induced rapamycin-independent component of mammalian target of rapamycin (Rictor) activation. A: confirmation of functional ITβ3 overexpression (ITβ3-OE) in vascular smooth muscle cells (VSMCs). Blots at the top and middle demonstrate ITβ3-green fluorescent protein (GFP) fusion protein expression (125 kDa) as well as light native ITβ3 expression (100 kDa) that was only detectable with higher exposure of blot membrane. The blot at the bottom shows β-tubulin loading control. The image on the right shows GFP-ITβ3 expression localized to the perinuclear region and focal adhesions as expected. B–D: Western blot analysis of in vitro effect of TGF-β1 stimulation on Rictor (B), Akt (C), and SMAD2 (D) phosphorylation in VSMCs (n = 5 per group) with or without the ITβ3 inhibitor SB-273005 (100 nM) or ITβ3-OE plated on regular plastic or vitronectin-coated cell culture dishes. Representative blots are shown for the ITβ3 inhibitor (left) and ITβ3-OE (right) data separately. Quantitative data for phospho-Rictor (pRictor) and pAkt are presented as the fold changes of TGF-β-stimulated (Stim) versus unstimulated (Unstim) intensity for each condition after normalization to total Rictor or total Akt protein. Data for pSMAD2 are presented as the ratio of pSMAD2 to total SMAD2 for the TGF-β-stimulated condition only. Phos, phosphorylated; WT, wild type. Groups were compared via one-way ANOVA followed by two-tailed, independent-samples pairwise post hoc t-tests. F-test P values are shown on each plot. *Post hoc comparison P < 0.05.

Article Snippet: Cells were plated on immobilized vitronectin (2 µg/ml, incubated 1 h and then removed, Promega, Madison, WI)-coated six-well plates at a density of 10 6 cells/well in complete DMEM (5% FBS).

Techniques: Activation Assay, Functional Assay, Over Expression, Expressing, Western Blot, In Vitro, Cell Culture, Two Tailed Test

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Proteomics reveals Rictor as a noncanonical TGF-β signaling target during aneurysm progression in Marfan mice

doi: 10.1152/ajpheart.00089.2018

Figure Lengend Snippet: Effect of β3-integrin (ITβ3) and vitronectin on metabolic and migration-proliferation vascular smooth muscle cell (VSMC) physiology. A: assessment of in vitro proliferation and migration by scratch assay of VSMCs under wild-type (WT), ITβ3-overexpressing (ITβ3-OE), and ITβ3 inhibition (Inhib) conditions (n = 3 per group). Quantification is reported as the percent cell-occupied area in scratch at 16 h relative to the cell-occupied area at 0-h transforming growth factor (TGF)-β stimulation. T, time. B: quantification of mitochondrial respiration in vitro in WT (line graph on the top left and box plots on the left side of each panel below) and ITβ3-OE (line graph on the top right and box plots on the right side of each panel below) VSMCs after 48 h of serum starvation in the presence or absence of TGF-β1 (5 ng/ml). The overall oxygen consumption rate (OCR) was measured using the Seahorse assay. Antimy./roten., antimycin-rotenone; ns, not significant. Groups were compared using two-tailed, independent-samples t-test. *P < 0.05; ^P < 0.05 relative to the WT-unstimulated condition; #P < 0.05 relative to the WT-TGF-β-stimulated condition.

Article Snippet: Cells were plated on immobilized vitronectin (2 µg/ml, incubated 1 h and then removed, Promega, Madison, WI)-coated six-well plates at a density of 10 6 cells/well in complete DMEM (5% FBS).

Techniques: Migration, In Vitro, Wound Healing Assay, Inhibition, Two Tailed Test