RT-qPCR analysis. HT-1080 cells were infected with SMAD2-specific shRNA lentiviral particles, total RNA was extracted from wild-type and knockdown cells, RT-qPCR was performed using gene-specific primers. ∆Ct (CtKD-CtWT) was used to calculate mRNA reduction (%) between wild-type and knockdown cells using the following formula: (1-1/2∆Ct) x 100%.

Western blotting analysis. SMAD2 protein expression in wild-type (WT) and shRNA knockdown (KD) HT-1080 cells was detected using Western blotting. Beta-TUBULINserved as a loading control. The blots were incubated with primary antibodies (Cat#69512, 1:5,000) against SMAD2 and Beta-TUBULIN, respectively, followed by incubating with HRP-conjugated goat anti-rabbit secondary antibody (Cat#201, 1:20,000). Images were developed using FeQ™ ECL Substrate Kit (Cat#226).