Journal: Journal of Clinical Medicine
Article Title: Tacrolimus Modulates TGF-β Signaling to Induce Epithelial-Mesenchymal Transition in Human Renal Proximal Tubule Epithelial Cells
Figure Lengend Snippet: The specific blockade of TβRI kinase ablated Smad2 activation and dramatically affected the induction of the EMT markers, vimentin and MMPs by FK506. HK-2 cells were cultured on 6-well plates and treated with vehicle control, 5 µM FK506 with or without 500 nM of a TGFβRI/ALK5 kinase inhibitor V (SD-208). ( A ) Cells were incubated for 48 h with SD-208 concentrations ranging from 0–20μM. LDH and resazurin assays were conducted to assess SD-208 inhibitor toxicity; ( B ) Whole cell protein was extracted at 48 h with RIPA buffer. Equal amounts of protein were separated by SDS-PAGE electrophoresis, transferred to nitrocellulose and indirectly probed for vimentin and fibronectin using a mAb and ECL detection system. Whole Smad2 and GAPDH was used as a loading control. A representative blot is shown from three independent experiments. Densitometry is shown for the replicate blots; ( C ) Cell culture supernatants were harvested, centrifuged to remove cellular debris and concentrated using Amicon Ultra-4 Centrifugal Filter Devices (Millipore). Gelatin substrate zymography was used to separate and assess MMP-9 and MMP-2 activity. Clear areas represent areas of gelatinolytic activity. A representative zymogram is shown from three independent experiments. ** and *** indicates different levels of significant compared to time-matched control; * , ** and *** indicates varying levels of significance compared to treatment + inhibitor.
Article Snippet: Gelatin Zymography: Cell supernatants were concentrated using Amicon Ultra-4 Centrifugal Filter Devices (Millipore, Billerica, MA, USA).
Techniques: Activation Assay, Cell Culture, Incubation, SDS Page, Electrophoresis, Zymography, Activity Assay