Figure 3.

Transduction of shRNAs using Adenovirus leads to efficient gene expression knockdown in primary SSc dermal fibroblasts. (A) Gene expression knockdown after shRNA transductions of SSc dermal fibroblasts. Plot shows the percentage of inhibition in gene expression for each shRNA in dermal fibroblasts from two different SSc donors (B) SSc dermal fibroblasts were transduced by shRNAs targeting GFP and mmNOS3 (negative controls) as well as 4 different shRNAs targeting ACTA2 or FN1. Crude conditions used only incubation with cell lysates from PerC5 cells that were used to produce the adenovirus. ASMA protein expression was measured using ASMA immunostaining after image quantification and normalization with the number of nuclei. EDA-Fibronectin protein expression was measured using an MSD assay. (C) Twelve SSc dermal fibroblasts (obtained from disease-affected skin or non-disease affected skin from 6 different SSc donors) were transduced with shRNAs targeting GFP (negative control) or ACTA2. Differences in ASMA protein expression between the GFP shRNA and the ACTA2 shRNA were determined using ASMA immunostaining after image quantification and normalization with the number of nuclei. Plot show both Z-primes and assay windows for each SSc dermal fibroblast tested (based on 120 data points for each donor and each shRNA).