Fig. 1: TGF-β downregulation and ROS generation by TGF-β shRNAs.

A Downregulation of TGF-β1 and TGF-β2 after infection with adenoviruses expressing TGF-β shRNAs. a Schematic structure of adenoviral vectors expressing TGF-β shRNA. dl324-∆E1A-∆E1B-∆E3-IX-U6-NC (Ad-NC) is a replication-incompetent adenovirus used as the negative control. It contains the scrambled DNA sequence for shRNA, which is under the control of the U6 promoter. dl324-∆E1A-∆E1B∆E3-IX-U6-shTGF-β1 or -β2 (Ad-shTGF-β1 or -β2) is a replication-incompetent adenovirus expressing human TGF-β1 or -β2 shRNA. NC negative control. b Downregulation of human transforming growth factor β1 (TGF-β1) or β2 by adenovirus expressing shTGF-β1 or -β2. Human A375 and HPAC cells were infected with various MOIs of adenovirus expressing shRNA targeting human TGF-β1 (Ad-shTGF-β1) or human TGF-β2 (Ad-shTGF-β2) or scrambled RNA (Ad-NC). TGF-β1 or -β2 mRNA was assayed by quantitative real-time polymerase chain reaction (qRT-PCR), and c protein levels were assayed by enzyme-linked immunosorbent assay (ELISA). MOI multiplicity of infection, NC negative control. Error bars represent the standard error from three independent experiments. B Various cancer cell lines were treated with adenovirus expressing shTGF-β1 or -β2 for 48 h and incubated for an additional 14 days for clonogenic assays. The numbers indicate the relative ratio of clone numbers to those of Ad-NC. C Global transcriptome analysis of TGF-β downregulation. Expression values of differentially expressed genes are shown in heat map format. Gene expression levels are visualized as row standardized z scores ranging from green (−1) to red (+1) across all samples. The rows are organized by hierarchical clustering analysis with complete linkage and Euclidean distance as a measure of similarity from samples of A375 cells (NC, shTGF-β1, shTGF-β2) (Left) and HPAC cells (NC, shTGF-β1, shTGF-β2) (Right). NC, A375, and HPAC cells infected with adenovirus expressing nonsense shRNA as a negative control at 100 MOI; shTGF-β1 or -β2; A375 and HPAC cells infected with adenoviruexpressing TGF-β1 or -β2 shRNA at 100 MOI. D Effect of adenovirus expressing shTGF-β1 or shTGF-β2 in various cancer cell lines. Each cell line was treated with adenovirus expressing shTGF-β1 or shTGF-β2 at 100 MOI. After 48 h, the expression levels of p-p38, p38, p-HSP27, HSP27, p-ERK, ERK, p-JNK, JNK, and GAPDH were detected via western blot analysis. E Pancreatic normal cell lines were treated with adenovirus expressing shTGF-β1 or -β2 at 100 MOI. After 48 h, the expression levels of p-p38, p38, p-HSP27, HSP27, p-ERK, ERK, p-JNK, JNK, and GAPDH were detected via western blot analysis. F ROS generation was induced by shTGF-β1- and shTGF-β2-expressing adenoviruses. A375 cells, HPAC cancer cells, and pancreatic normal cells were infected with adenovirus expressing shTGF-β1 or -β2 at 100 MOI, respectively, after 48 h of incubation with DCF-DA (20 μM, 1 h) for the detection of ROS using a fluorescent reader and microscopy. Cell viability was tested via an MTS viability assay after 48 h of infection. Error bars represent the standard error from three independent experiments. p Values <0.01 indicate a very significantly different viability of Ad-shTGF-β1 or -β2 compared to the control (Ad-NC). G Effects of NAC treatment with adenovirus expressing TGF-β1 or -β2 shRNA in melanoma and pancreatic cancer cells. A375 (top) or HPAC (bottom) cells were infected with adenovirus expressing shTGF-β1 or -β2 at 100 MOI, respectively. After 6 h, infected cells were treated with NAC (10 mM) for 42 h and then incubated with DCF-DA (20 μM, 1 h) for the detection of ROS using a fluorescent reader and microscopy. H A375 and HPAC cells were infected with adenovirus expressing shTGF-β1 or -β2. After 6 h, infected cells were treated with NAC (10 mM) for 42 h, and then the expression levels of PARP, p-ASK1, ASK1, p-p38, p38, p-JNK, JNK, Trx, and GAPDH were detected by western blot analysis