Fig 3.TIF (2.15 MB)

ASS1 protein is upregulated in 3D in mesothelioma cells but not in normal mesothelial cells.

figure

posted on 2016-03-16, 00:08 authored by Dario Barbone, Loes Van Dam, Carlo Follo, Puthen V. Jithesh, Shu-Dong Zhang, William G. Richards, Raphael Bueno, Dean A. Fennell, V. Courtney Broaddus

(A) ASS1 antibodies (BD clone 25, Sigma clone 2B10, and Millipore clone 2C10) were tested against ASS1-positive MSTO pIRES-ASS1 (as a positive control) and ASS1-negative MSTO pIRES-EV (as a negative control). Monoclonal antibody clone 2C10 provides the most accurate detection of ASS1 protein levels. (B) Immunoblot analysis of ASS1, ANXA4 and MVP in monolayers (m) and spheroids (s) grown from 6 mesothelioma cell lines (M28, SARC, VAMT, REN, JMN) and MSTO-211H (known to be ASS1-negative). MSTO pIRES-ASS1 cells were used as positive control for ASS1. ASS1 was the most consistently upregulated in 3D among the panel of cells tested. JMN and MSTO cells showed no ASS1 expression. (C) Immunoblot analysis of ASS1 in normal mesothelial cells grown as monolayers and spheroids. ASS1-negative MSTO pIRES-EV and ASS1-positive MSTO pIRES-ASS1 were used as controls for ASS1. Normal mesothelial cells did not upregulate ASS1 when grown in 3D.