Smad2 and Smad3 Play Different Roles in Rat Hepatic Stellate Cell Function and -Smooth Muscle Actin Organization

Supplemental Material

This article contains the following supporting material:

• Video 1 - Smad3-overexpressing cells demonstrate different spreading behavior-1
  
  Smad3-overexpressing cells demonstrate different spreading behavior. Activated stellate cells (day 7 after isolation) were infected with Ad-ΒGal (Video1.mov) or Ad-wt3 (Video2.mov). Cells were trypsinized and replated 24h after infection. Time-lapse images of both cell types were taken for up to 16 hours after replating as described in Materials and Methods. Note the numbers of membrane protrusions and the rapidly changing cell outline in the Ad-ΒGal-infected cells compared to the Ad-wt3-infected cells.
• Video 2 - Smad3-overexpressing cells demonstrate different spreading behavior-2
• Video 3 - Smad3-overexpressing cells migrate through Matrigel ™.
  
  HSC plated on Matrigel ™ and infected at day 2 after isolation with Ad-wt3 were fixed and stained at day 7 with an antibody against α-SMA (red) and with DAPI (blue). Fluorescent images were acquired from the top to the bottom of the layer of Matrigel ™as a Z-series (2 ??m steps) and converted to a movie using IPLab ™ 3.9 software (Scanalytics). The Smad3-overexpressing HSC have migrated through the Matrigel ™ and begun to spread on the underlying support and to express α-SMA.
• Figure S1 - Smad3 is the dominant Smad determining HSC phenotype.
  
  HSC were infected with combinations of two different adenoviruses at day 2 after isolation, as indicated. Light microscopy of these cells at day 7 demonstrated that HSC infected with Ad-wt3 (upper panels) are phenotypically different than HSC without Ad-wt3 (lower panels), regardless of the co-infected adenovirus. Bar, 50 μm.