Fig 1: Twist1 binding region sequences are associated with genes in the Wnt signaling pathway. A. Wnt pathway genes associated with Twist1 E-box-containing binding regions detected by ChIP-seq in E12.5 ECCs include receptor tyrosine kinase-like orphan receptor 2 (Ror2-b), homeodomain interacting protein kinase 2 (Hipk2), secreted frizzled-related protein 1 (Sfrp1), frizzled homolog 4 (fzd4), and frizzled homolog 8 (fzd8). Twist1 binding to these sequences was confirmed by ChIP with anti-Twist1 in E12.5 ECCs followed by quantification by qPCR. B. Wnt pathway genes associated with Twist1 E-box containing binding regions detected by ChIP-seq in E10.5 limb buds include Wnt5b, lymphoid enhancer binding factor 1 (Lef1), nemo like kinase (Nlk), casein kinase 1, epsilon (Csnk1e), disabled 2 interacting protein (Dab2ip), mutated in colorectal cancers (Mcc), and frizzled homolog 3 (Fzd3). Twist1 binding to these sequences was confirmed by ChIP with anti-Twist1 in E10.5 limb bud followed by quantification by qPCR. All ChIP experiments were quantified with qPCR, and fold-enrichment was calculated relative to normal rabbit IgG control. All analyses were performed in biological triplicate. * indicates p < 0.05 as determined by student’s t-test comparing IgG and Twist1 ChIP qPCR ΔΔCt values.
Fig 2: Characterization of VIC phenotypes in remodeling mitral heart valve structures. Immunohistochemistry to show expression of smooth muscle a-actin (SMA) with Periostin (A,A’), Twist1 (B,B’) and Vimentin (C,C’) in cells within the elongating mitral valve leaflet (mural). Arrows indicate SMA expression (red) and arrowheads indicate Periostin (A,A’) Twist1 (B,B’) and Vimentin (C,C’) shown in green. The boxed areas in A-C are shown at higher magnification in (A’–C’).
Fig 3: Characterization of VIC phenotypes during early heart valve development. Immunohistochemistry to show expression of smooth muscle α-actin (SMA) with Periostin (A,A’,D), Twist1 (B,B’,E) and Vimentin (C,C’,F) in cells within the inferior and superior endocardial cushions at E12.5 (A–C’) and atrioventricular valve primordia at E13.5 (D–F). Arrows indicate SMA expression (red) and arrowheads indicate Periostin (A,A’,D) Twist1 (B,B’,E) and Vimentin (C,C’,F) shown in green. The boxed areas in A-C are shown at higher magnification in (A’–C’). The white lines highlight the developing valve regions. I, inferior cushion; IVS, intraventricular septum; LV, left ventricle; P, parietal leaflets; S, superior cushion.
Fig 4: Resveratrol attenuates the upregulation of Hedgehog/EMT signaling induced by LPA. SKOV3 (A), OVCAR3 (B) and OAW42 (C) human ovarian cancer cells were plated on Petri dishes and let grow to confluence. Cells were exposed for 72 h to 100 µM resveratrol (RV) or 20 µM LPA or both simultaneously. Cell homogenates were analyzed by Western blotting for the expression of GLI1, BMI-1, SNAIL-1 and TWIST1. The membranes were stripped and re-probed with different antibodies. The β-actin for OVCAR3 and OAW42 correspond to the ones reported in Figure 4. Densitometric data (normalized on the loading control β-actin) are reported. All blots are representative of three independent experiments.
Fig 5: Expression and release of IL-6, MCP-1, and adiponectin into conditioned media after siRNA treatment of cultured adipocytes. Human in vitro–differentiated adipocytes were treated with 50 nmol/l control siRNA or siRNA directed against twist1. Expression of the inflammatory genes IL-6, TNF-a, and MCP-1 as well as adiponectin was measured after siRNA treatment (A). mRNA levels were normalized to the reference gene GAPDH. Release of IL-6 (B), MCP-1 (C), and adiponectin (D) was measured in conditioned media; n = 4 (IL-6) and n = 3 (MCP-1/adiponectin). Results shown are mean ± SEM and mRNA levels are shown as relative expression (fold change to control siRNA). **P < 0.01, *P < 0.05.
Supplier Page from MilliporeSigma for Anti-Twist1 antibody produced in rabbit