Twist1 Antibody - #AF5224

Fig 1 TGF-β1 promotes cell motility via accelerated ΔNp63α proteasomal degradation independent of Smad pathway. (A) MCF-10A cells were infected with lentivirus expressing ΔNp63α or a vector control and were selected for drug resistance. The stable cells were then treated with vehicle or TGF-β1 (10 ng/mL) for 48 h or 72 h prior to examination of cell morphology. Representative images were shown. Scale bar = 100 μm. (B, C) MCF-10A cells were treated with an indicated dose of TGF-β1 for 12 h or were treated with 10 ng/ml TGF-β1 for an indicated time, followed by western blot analyses. (D–G) MCF-10A cells stably expressing HA-TβRI were infected with lentivirus expressing Flag-ΔNp63α or a vector control, followed by (D) western blot analyses, (E) morphology assays, or (F, G) transwell assays. Data from 3 independent experiments in triplicates were presented as means ± SD. Scale bar = 100 μm. ***p < 0.001. (H) MCF-10A cells were grown in the presence of 10 ng/ml TGF-β1 for 36 h and then treated with 10 μM phosphorylated Smad3 inhibitor SIS3 for 8 h prior to western blot analyses. (I) MCF-10A stable cells expressing HA-TβRI were infected with lentivirus expressing HA-Smad7 or a vector control, followed by western blot analyses. (J) MCF-10A cells were grown in the presence of 10 ng/ml TGF-β1 for 36 h and then treated with 10 μM MEK inhibitor U0126 for 10 h prior to western blot analyses. (K) MCF-10A cells stably expressing HA-TβRI were treated with CHX (50 μg/mL) for an indicated time interval and then subjected to western blot analyses. The ΔNp63α protein levels were quantified and presented. (L) MCF-10A cells were grown in the presence of 10 ng/ml TGF-β1 for 36 h and then treated with 10 μM MG132 for 6 h or 45 μM CLQ for 12 h prior to western blot analyses. The underlying data for this figure can be found in S1 Data. CHX, cycloheximide; CLQ, chloroquine; TGF-β, transforming growth factor-β.

Fig 2. FBXO3 promotes cell migration and tumor metastasis via up-regulation of Twist1 expression. (A) MDA-MB-231 or Hs578T cells stably expressing shFBXO3-#1, shFBXO3-#2, or shGFP (-) were subjected to western blot analyses. (B) MDA-MB-231 or MCF-10A cells stably expressing HA-FBXO3 WT, HA-FBXO3ΔF (ΔF), or a vector control (-) were subjected to western blot analyses. (C) MDA-MB-231 cells stably expressing HA-FBXO3 were infected with a recombinant lentivirus expressing specific shRNA targeting to Twist1 or a vector control (-), and were subjected to western blot analyses. (D) MDA-MB-231 stable cells were subjected to transwell assays. Data from 3 independent experiments were presented as means ± SD. ***p < 0.001. Scale bar = 100 μm. (E–G) MDA-MB-231 stable cells were subjected to tail vein injection in BALB/c nude mice (n = 5). Lungs of mice were photographed (E) and stained with HE staining (F), and the number of tumors per mouse was counted (G). Data were presented as means ± SEM. *p < 0.05. (H, I) Consecutive TMA slides derived from human breast cancer specimens (HBreD030PG03, OUTDO, Shanghai, China) were subjected to IHC for expression of FBXO3 and Twist1. IHC staining was quantified by AOD. Representative images of IHC staining and Pearson correlation of FBXO3 and Twist1 expression were shown. Scale bar = 100 μm. The data underlying the graphs shown in the figure can be found in S1 Data. AOD, average optical density; IHC, immunohistochemistry; TMA, tissue microarray; WT, wild-type.